Morphine and morphine precursors

ABSTRACT

Methods and materials related to the use of morphine, morphine precursors (e.g., reticuline), and inhibitors of morphine synthesis or activity to treat diseases, to reduce inflammation, or to restore normal function are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/576,448, filed Aug. 31, 2007, which is a U.S. National Stageapplication under 35 U.S.C. §371 and claims benefit under 35 U.S.C.§119(a) of International Application No. PCT/US2005/035628 having anInternational Filing Date of Sep. 30, 2005, which claims the benefit ofU.S. Provisional Application Ser. No. 60/714,769, having a filing dateof Sep. 6, 2005, and U.S. Provisional Application Ser. No. 60/615,048having a filing date of Oct. 1, 2004. The disclosures of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was provided by the federalgovernment, which may have certain rights in the invention.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in usingmorphine, morphine precursors (e.g., reticuline),morphine-6β-glucuronide, and inhibitors of morphine synthesis oractivity to treat mammals.

2. Background Information

Many people suffer from conditions such as depression, neurodegenerativediseases (e.g., Alzheimer's disease), pro-inflammatory diseases,autoimmune disorders, and atherosclerosis. In many cases, few, if any,successful treatments are available for these people.

Morphine is a powerful analgesic that is routinely used to reduce painin humans. For example, surgery patients are typically instructed totake 5 to 10 mg of morphine per person to alleviate pain caused by thesurgical procedure. In some cases, patients suffering from extreme pain(e.g., burn victims or cancer patients) are instructed to take higherdoses of morphine. For moderate to severe pain, the optimalintramuscular dosage is considered to be 10 mg per 70 kg body weightevery four hours. The typical dose range is from 5 to 20 mg every fourhours, depending on the severity of the pain. The oral dose range isbetween 8 and 20 mg, but orally administered morphine has substantiallyless analgesic potency. Orally administered morphine can exhibit aboutone-tenth of the effect produced by subcutaneous injection of morphinesince orally administered morphine is rapidly destroyed as it passesthrough the liver after absorption. The intravenous route is usedprimarily for severe post-operative pain or in an emergency. In suchcases, the dose range is between 4 and 10 mg, and the analgesic effectensues almost immediately.

SUMMARY

This document provides methods and materials related to using morphine,morphine-6β-glucuronide, morphine precursors (e.g., reticuline),inhibitors of morphine synthesis or activity, and inhibitors of dopaminesynthesis to treat diseases, to reduce inflammation, or to restorenormal function. For example, this document provides compositionscontaining morphine, morphine-6β-glucuronide, morphine precursors,inhibitors of morphine synthesis, inhibitors of morphine activity,inhibitors of dopamine synthesis, or combinations thereof. This documentalso provides methods for using such compositions (e.g., method forproviding a mammal with a long-term, low level of morphine). Asdescribed herein, a long-term, low level of morphine can be achieved ina mammal by repeatedly administering a low dose of morphine, byrepeatedly administering a morphine precursor, or by repeatedlyadministering a combination of morphine and morphine precursors. In somecases, inhibitors such as dopamine β-hydroxylase inhibitors can be usedto inhibit the dopamine to norepinephrine step in adrenaline synthesis,which can result in an endogenous dopamine level increase as well as anendogenous morphine level increase.

Providing a mammal with a long-term, low level of morphine can allow themammal to experience behavioral changes (e.g., a general overall calmfeeling). In addition, providing a mammal with a long-term, low level ofmorphine can allow the mammal to experience reduced inflammatoryresponses and can allow the mammal to maintain an increased, basal levelof constitutive nitric oxide release. In some cases, the compositionsprovided herein can be used to down regulate immune, vascular, neural,and gastrointestinal tissues via nitric oxide produced within a mammal.For example, the compositions provided herein can be used to reduce theexcited state of inflamed gastrointestinal tissues in mammals havingCrohn's disease.

The methods and materials provided herein also can be used to treat(e.g., reduce the severity of symptoms) neural conditions (e.g.,schizophrenia, chronic pain, mania, depression, psychosis, paranoia,autism, stress, Alzheimer's disease, or Parkinson's disease), immuneconditions (e.g., pro-inflammatory diseases, autoimmune disorders,histolytic medullary reticulosis, lupus, or arthritis), vascularconditions (e.g., atherosclerosis or neuronal vasculopathy),gastrointestinal conditions (e.g., colitis, Crohn's disease, orirritable bowel syndrome), or addiction (e.g., opiate addiction). Forexample, morphine or a morphine precursor such as reticuline,norlaudanosoline, L-DOPA, or codeine can be used to treat neuralconditions such as neurovascular alterations involving hypothalamichormone secretion (e.g., reproductive and growth hormones).

As disclosed herein, prolonged treatment with a low dose of morphine canresult the continued positive effects of morphine such as nitric oxiderelease, without the need to escalate morphine dosages with time toachieve the same beneficial effects. In addition, the use of low dosesof morphine can allow patients to experience the beneficial effects ofmorphine, while not experiencing possible negative effects of morphine(e.g., addiction or powerful analgesia). Likewise, treating patientswith a morphine precursor such as reticuline can allow patients toexperience the beneficial effects of morphine, while not experiencingpossible negative effects of morphine (e.g., addiction or powerfulanalgesia). For example, using a morphine precursor such as reticulinecan allow patients to receive a low dose of morphine indirectly with areduced risk of overdosing.

In general, one aspect of this document features a method for inducingnitric oxide release from cells in a mammal. The method comprises, orconsists essentially of, administering, to the mammal, a composition inan amount, at a frequency more frequent than once a week, and for aduration longer than one month, wherein the composition comprises, orconsists essentially of, morphine or morphine-6β-glucuronide, andwherein the amount of the composition results in less than 0.05 mg ofthe morphine or morphine-6β-glucuronide being administered to the mammalper kg of body weight of the mammal per day. The cells can be immunecells. The mammal can be a human. The composition can contain a morphineprecursor. The composition can be in the form of a tablet. Thecomposition can contain selenium. The composition can contain arginine(e.g., L-arginine). The composition can contain a calcium source. Theamount of the composition can result in less than 0.025 mg of themorphine or morphine-6β-glucuronide being administered to the mammal perkg of body weight of the mammal per day. The e amount of the compositioncan result in less than 0.01 mg of the morphine ormorphine-6β-glucuronide being administered to the mammal per kg of bodyweight of the mammal per day. The frequency can be more frequent thanfour times a week. The frequency can be between two and five times aday. The frequency can be once a day. The duration can be longer thantwo months. The duration can be longer than three months. Thecomposition can contain morphine. The composition can containmorphine-6β-glucuronide. The composition can contain morphine andmorphine-6β-glucuronide.

In another embodiment, this document features a method for inducingnitric oxide release from cells in a mammal. The method comprises, orconsists essentially of, administering, to the mammal, a composition inan amount, at a frequency more frequent than once a week, and for aduration longer than one month, wherein the composition comprisesthebaine or codeine, and wherein the amount of the composition resultsin less than 0.05 mg of the thebaine or codeine being administered tothe mammal per kg of body weight of the mammal per day. The cells can beimmune cells. The mammal can be a human. The composition can containmorphine in an amount that results in less than 0.05 mg of the morphinebeing administered to the mammal per kg of body weight of the mammal perday. The composition can be in the form of a tablet. The composition cancontain selenium. The composition can contain arginine (e.g.,L-arginine). The composition can contain a calcium source. The amount ofthe composition can result in less than 0.01 mg of the thebaine orcodeine being administered to the mammal per kg of body weight of themammal per day. The amount of the composition can result in less than0.005 mg of the thebaine or codeine being administered to the mammal perkg of body weight of the mammal per day. The frequency can be morefrequent than four times a week. The frequency can be between two andfive times a day. The frequency can be once a day. The duration can belonger than two months. The duration can be longer than three months.The composition can contain thebaine. The composition can containcodeine. The composition can contain thebaine and codeine.

In another embodiment, this document features a method for inducingnitric oxide release from cells in a mammal. The method comprises, orconsists essentially of, administering, to the mammal, a composition inan amount, at a frequency more frequent than once a week, and for aduration longer than one month, wherein the composition comprises one ormore agents selected from the group consisting of reticuline,norlaudanosoline, and salutaridine, and wherein the amount of thecomposition results in less than 1 mg of the one or more agents beingadministered to the mammal per kg of body weight of the mammal per day.The cells can be immune cells. The mammal can be a human. Thecomposition can contain morphine in an amount that results in less than0.05 mg of the morphine being administered to the mammal per kg of bodyweight of the mammal per day. The composition can be in the form of atablet. The composition can contain selenium. The composition cancontain arginine (e.g., L-arginine). The composition can contain acalcium source. The amount of the composition can result in less than0.5 mg of the one or more agents being administered to the mammal per kgof body weight of the mammal per day. The amount of the composition canresult in less than 0.05 mg of the one or more agents being administeredto the mammal per kg of body weight of the mammal per day. The frequencycan be more frequent than four times a week. The frequency can bebetween two and five times a day. The frequency can be once a day. Theduration can be longer than two months. The duration can be longer thanthree months. The composition can contain reticuline, norlaudanosoline,and salutaridine.

In another embodiment, this document features a method for inducingnitric oxide release from cells in a mammal. The method comprises, orconsists essentially of, administering, to the mammal, a composition inan amount, at a frequency more frequent than once a week, and for aduration longer than one month, wherein the composition comprisesdopamine or L-DOPA, and wherein the amount of the composition results inless than 1 μg of the dopamine or L-DOPA being administered to themammal per kg of body weight of the mammal per day. The cells can beimmune cells. The mammal can be a human. The composition can containmorphine in an amount that results in less than 0.05 mg of the morphinebeing administered to the mammal per kg of body weight of the mammal perday. The composition can be in the form of a tablet. The composition cancontain selenium. The composition can contain arginine (e.g.,L-arginine). The composition can contain a calcium source. The amount ofthe composition can result in less than 0.5 mg of the one or more agentsbeing administered to the mammal per kg of body weight of the mammal perday. The amount of the composition can result in less than 0.05 mg ofthe one or more agents being administered to the mammal per kg of bodyweight of the mammal per day. The frequency can be more frequent thanfour times a week. The frequency can be between two and five times aday. The frequency can be once a day. The duration can be longer thantwo months. The duration can be longer than three months. Thecomposition can contain reticuline, norlaudanosoline, and salutaridine.

In another aspect, this document features a composition comprising, orconsisting essentially of, morphine and selenium. The composition cancontain between 35 μg and 700 μg of morphine. The composition cancontain between 55 μg and 300 μg of selenium. The composition cancontain arginine (e.g., L-arginine). The composition comprises between 1mg and 500 mg of arginine. The composition can contain a calcium source.The composition can contain between 250 μg and 1.5 g (e.g., between 1 gand 1.3 g) of the calcium source. The calcium source can be calciumcitrate. The composition can contain one or more agents selected fromthe group consisting of tyrosine, tyramine, phenylalanine, 3,4dihydroxyphenyl pyruvate, dihydroxyphenyl acetaldehyde, dopamine,L-DOPA, reticuline, norlaudanosoline, salutaridine, thebaine, andcodeine. The composition can contain one or more agents selected fromthe group consisting of CYP2D6 and CYP2D7 inhibitors.

In another embodiment, this document features a composition comprising,or consisting essentially of, morphine and arginine (e.g., L-arginine).The composition can contain between 35 μg and 700 μg of morphine. Thecomposition can contain between 1 mg and 500 mg of arginine. Thecomposition can contain a calcium source. The composition can containbetween 250 μg and 1.5 g (e.g., between 1 g and 1.3 g) of the calciumsource. The calcium source can be calcium citrate. The composition cancontain one or more agents selected from the group consisting oftyrosine, tyramine, phenylalanine, 3,4 dihydroxyphenyl pyruvate,dihydroxyphenyl acetaldehyde, dopamine, L-DOPA, reticuline,norlaudanosoline, salutaridine, thebaine, and codeine. The compositioncan contain one or more agents selected from the group consisting ofCYP2D6 and CYP2D7 inhibitors.

In another embodiment, this document features a composition for reducingthe level of morphine produced in cells, wherein the compositioncomprises, or consists essentially of, L-DOPA and dopamine. Thecomposition can contain between 25 mg and 500 mg (e.g., 250 mg) ofL-DOPA. The composition can contain between 25 mg and 500 mg (e.g., 250mg) of dopamine The composition can contain an equal amount of L-DOPAand dopamine.

In another aspect, this document features a method for increasingproduction of morphine in a mammal. The method comprises, or consistsessentially of, administering a composition to the mammal underconditions effective to increase the amount of morphine produced bycells within the mammal, wherein the composition comprises one or moreagents selected from the group consisting of reticuline,norlaudanosoline, salutaridine, thebaine, and codeine. The cells can beimmune cells. The mammal can be a human. The composition can containmorphine. The composition can be in the form of a tablet. Thecomposition can contain selenium. The composition can contain arginine(e.g., L-arginine). The composition can contain a calcium source. Themethod can include, prior to the administering step, identifying themammal as needing an increase in morphine. The method can include, afterthe administering step, monitoring the mammal to confirm an increase inmorphine. The method can include administering the composition to themammal at a frequency more frequent than once a month. The method caninclude administering the composition to the mammal at a frequency morefrequent than once a week. The method can include administering thecomposition to the mammal at a frequency between once and five times aday. The method can include administering the composition to the mammalat a frequency more frequent than once a week and for a duration longerthan one month. The duration can be longer than three months.

In another embodiment, this document features a method for treating amammal having a condition selected from the group consisting ofschizophrenia, mania, depression, psychosis, chronic pain, paranoia,autism, stress, Alzheimer's disease, Parkinson's disease,pro-inflammatory diseases, autoimmune disorders, histolytic medullaryreticulosis, lupus, arthritis, atherosclerosis, neuronal vasculopathy,gastrointestinal conditions, and addiction. The method comprises, orconsists essentially of, administering a composition to the mammal underconditions wherein the severity of a symptom of the condition isreduced, wherein the composition comprises one or more agents selectedfrom the group consisting of reticuline, norlaudanosoline, salutaridine,thebaine, and codeine. The method can include, prior to theadministering step, identifying the mammal as having the condition. Themethod can include, after the administering step, monitoring the mammalto confirm an reduction is the severity. The mammal can be a human. Thecomposition can be administered orally. The composition can beadministered to the mammal in an amount such that the mammal receivesbetween about 1 and 5 mg of at least one of the one or more agents perkg body weight of the mammal. The composition can be administered to themammal at a frequency more frequent than once a month. The compositioncan be administered to the mammal at a frequency between once and 5times a day or week.

In another embodiment, this document features a method for treating amammal having a condition selected from the group consisting ofschizophrenia, mania, depression, psychosis, chronic pain, paranoia,autism, stress, Alzheimer's disease, Parkinson's disease,pro-inflammatory diseases, autoimmune disorders, histolytic medullaryreticulosis, lupus, arthritis, atherosclerosis, neuronal vasculopathy,gastrointestinal conditions, and addiction. The method comprises, orconsists essentially of, administering, to the mammal, a composition inan amount, at a frequency more frequent than once a week, and for aduration longer than one month, wherein the composition comprisesmorphine or morphine-6β-glucuronide, and wherein the amount of thecomposition results in less than 0.05 mg of the morphine ormorphine-6β-glucuronide being administered to the mammal per kg of bodyweight of the mammal per day. The method can include, prior to theadministering step, identifying the mammal as having the condition. Theseverity of a symptom of the condition can be reduced at a time point atleast one month following an initial administration of the composition.The method can include, after the administering step, evaluating themammal to confirm a reduction in the severity of a symptom of thecondition. The mammal can be a human. The composition can beadministered orally. The composition can be administered to the mammalat a frequency more frequent than once a week. The composition can beadministered to the mammal at a frequency between once and 5 times a dayor week. The composition can contain morphine. The composition cancontain morphine-6β-glucuronide. The composition can contain morphineand morphine-6β-glucuronide.

In another aspect, this document features a dietary supplementcomprising, or consisting essentially of, selenium, morphine, andarginine (e.g., L-arginine). The composition can contain any of theadditional components described herein such as a morphine precursor or acalcium source.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is an HPLC chromatogram of ganglia extraction. The topchromatogram of ganglia incubated with 0.5 μg of reticuline for 1 hourdemonstrates a level of 5 ng/mg morphine tissue wet weight. The middlechromatogram is for control ganglia. The bottom chromatogram is for amorphine standard (15 ng).

FIG. 2 is a graph plotting morphine levels for the amount of reticulineincubated with Mytilus edulis ganglia. Ganglia were incubated with 1.0,10, 50, or 100 ng (per ganglion) of reticuline for 60 minutes. Morphineconcentrations were obtained by RIA. One Way ANOVA analysis demonstratedthat the morphine levels in ganglion incubated with reticuline weresignificantly higher than control at 50 and 100 ng of reticuline. Oneganglion weighs about 1.7 mg.

FIG. 3 is a graph plotting morphine levels versus the time Mytilusedulis ganglia were incubated with reticuline (50 ng/ganglion). Theresults of morphine concentration were obtained by RIA. One Way ANOVAanalysis demonstrated that the morphine level in ganglia incubated withreticuline was significantly higher than control at 60 minutes.

FIG. 4 is a graph plotting NO release versus time for ganglia treatedwith reticuline (10⁻⁷ M) alone, morphine (10⁻⁶ M) alone, or naloxone(10⁻⁶ M) plus reticuline (10⁻⁷ M).

FIG. 5 is a graph plotting morphine levels detected for Mytilus edulispedal ganglia treated in vitro with 1.0, 10, 50, or 100 ng of L-dopa orreticuline for 60 minutes. Vehicle treated ganglia morphine levelsbefore and after the incubation with the respective precursors wasdetermined (2.1±0.44 and 2.3±0.31 ng/ganglia, respectively). One WayANOVA analysis revealed that the morphine levels in ganglion incubatedwith reticuline (50 and 100 ng) or L-DOPA (50 and 100 ng) weresignificantly higher than those measured in control ganglion (P<0.01 for50 ng amounts, and P<0.001 for 100 ng amounts). One ganglion weighsabout 1.7 mg. All determinations were replicated four times, and themean±SEM is presented.

FIG. 6 is a graph plotting morphine levels versus the time Mytilusedulis ganglia were incubated with reticuline or L-DOPA (1 μg/10ganglia). One Way ANOVA analysis revealed that the ganglionic morphinelevels in ganglia incubated with the morphine precursors weresignificantly higher than control at 30 and 60 minutes (P<0.01). Alldeterminations were replicated four times, and the mean graphed±SEM.

FIG. 7 is a bar graph plotting the level of morphine (ng/ganglion) inanimals one hour after receiving an injection of reticuline or L-DOPA (1μg/animal) into the base of Mytilus foot. Morphine levels weresignificantly increased compared to control levels (P<0.01; One WayANOVA analysis). All determinations were replicated four times, and themean±SEM is presented.

FIG. 8 is a graph plotting morphine levels detected for Mytilus edulispedal ganglia treated in vitro with 1.0, 10, 50, or 100 ng ofnorlaudanosoline for 60 minutes. One Way ANOVA analysis revealed thatthe morphine levels in ganglion incubated with norlaudanosoline weresignificantly higher than control at 50 and 100 ng of norlaudanosoline.All determinations were replicated four times, and the mean graphed±SEM.

FIG. 9 is a graph plotting morphine levels versus the time Mytilusedulis ganglia were incubated with norlaudanosoline (100 ng/ganglia).One Way ANOVA analysis revealed that the morphine levels in gangliaincubated with norlaudanosoline were significantly higher than controlsat 30 and 60 minutes (P<0.01). All determinations were replicated fourtimes, and the mean graphed±SEM.

FIG. 10 is a Q-TOF analysis of authentic morphine extracted from HPLCfractions (inset). WBC morphine exhibited the same MS as authenticmaterial.

FIG. 11A is a graph plotting the amount of morphine produced from humanWBC obtained from a Buffy coat and incubated with the indicated amountof tyramine for one hour (P<0.001, One Way ANOVA at the 10⁻⁷ to 10⁻⁶ Mconcentrations). FIG. 11B contains bar graphs plotting the amount ofmorphine produced from human WBC obtained from a Buffy coat andincubated with the indicated amount of norlaudanosoline (THP),reticuline, or L-DOPA for one hour (P<0.001, One Way ANOVA at the 10⁻⁷to 10⁻⁶ M concentrations). Each experiment was repeated three times, andthe mean±SEM is presented.

FIG. 12 is a graph plotting the amount of morphine produced from humanPMNs obtained from a Buffy coat and incubated with tyramine (T; 10⁻⁶ M)and the indicated amount of bufuralol. The tyramine-induced morphinelevels were diminished significantly with increasing concentrations ofbufuralol (P<0.001, One Way ANOVA). Each experiment was repeated threetimes, and the mean±SEM is presented.

FIG. 13 is a graph plotting the amount of morphine produced from humanPMNs obtained from a Buffy coat and incubated with tyramine (T; 10⁻⁶ M),norlaudanosoline (THP; 10⁻⁷M), or codeine together with either quinidine(10⁻⁶ M) or paroxetine (10⁻⁶ M). The tyramine- and THP-induced morphinelevels were diminished by treatment with quinidine (P<0.001, One WayANOVA compared to tyramine and THP stimulated morphine levels,respectively). Each experiment was repeated five times, and the mean±SEMis presented.

FIG. 14 is a graph plotting the level of PMN activation for cellstreated as follows: 1, PMN activity level after 60 minutes of notreatment; 2, PMNs incubated with IL-1β; 3, PMNs incubated with L-DOPA(10⁻⁶ M); 4, mixed culture with 50% L-DOPA exposed cells and 50% IL-1βexposed cells for one hour; 5, mixed culture with 50% L-DOPA exposedcells and 50% IL-1β exposed cells for one hour (the IL-1β exposed cellswere exposed to naloxone (10⁻⁶ M) five minutes before being added to themixed culture). Cells mixed without treatment from the two groupsexhibited only a 6% increase over that of their respective controls.Each experiment was replicated four times, and the mean±SEM ispresented.

FIG. 15 is a diagram of the biosynthetic pathways for producing morphineand catecholamines.

FIG. 16A is a graph plotting morphine levels in Mytilus edulis gangliatreated with the indicated amount of tyrosine or tyramine for 60minutes. At concentrations of 10⁻⁷ and 10⁻⁶ M, the mean values werestatistically significant (P<0.001) as compared to untreated ganglia.FIG. 16B is a graph plotting morphine levels versus the time Mytilusedulis ganglia were incubated with tyrosine or tyramine (10⁻⁶M). At 45-and 60-minute incubations, the mean values were statisticallysignificant (P<0.001) as compared to untreated ganglia. Alldeterminations were replicated three times, and the mean graphed±SEM.

FIG. 17 is a graph plotting the amount of morphine produced from Mytilusedulis ganglia incubated with tyramine (T; 10⁻⁶ M) and the indicatedamount of quinidine. The tyramine-induced morphine levels werediminished significantly with increasing concentrations of quinidine(P<0.001, One Way ANOVA). Each experiment was repeated five times, andthe mean±SEM is presented.

FIG. 18 is a graph plotting the amount of morphine produced from Mytilusedulis ganglia incubated with tyrosine (T; 10⁻⁶ M) and the indicatedamount of alpha-methyl-para-tyrosine (AMPT). The tyrosine-inducedmorphine levels were diminished significantly with increasingconcentrations of AMPT (P<0.001, One Way ANOVA). Each experiment wasrepeated four times, and the mean±SEM is presented.

FIG. 19 contains representative HPLC chromatograms demonstratingganglionic and hemolymph dopamine (DA) levels can be modulated bytyramine and quinidine (10⁻⁶ M) exposure. Ganglia, Panel A: Tyramineinjection (100 μg/animal, under foot) resulted in 9.17±1.21 μg of DA/g.Ganglia, Panel B: Tyramine and quinidine injections (100 μg/animal)resulted in 2.57±0.32 μg of DA/g. Ganglia, Panel C: PBS injectionresulted in 4.78±0.27 μg of DA/g. Hemolymph, Panel A: PBS incubationresulted in 10.13±0.34 μg of DA/mL. Hemolymph, Panel B: Tyramine (100μg/mL) and quinidine (10 μg/mL) exposure to pedal ganglia resulted in10.24 μg of DA/mL. Hemolymph, Panel C: Tyramine (100 μg/mL) incubationresulted in 16.47±2.28 μg of DA/mL.

FIG. 20A is a graph plotting the level of DA detected in ganglia orhemolymph from untreated animals or animals treated with tyramine (10⁻⁶M) or tyramine (10⁻⁶ M) plus quinidine (10⁻⁶ M). Quinidine blocked theincrease in endogenous ganglionic and hemolymph DA levels caused by theexposure of the pedal ganglia to tyramine alone (P<0.001). FIG. 20B is agraph plotting the level of morphine detected in ganglia from untreatedanimals or animals treated with codiene (10⁻⁶ M) or codiene plusquinidine (10⁻⁶ M). Quinidine blocked the increase in endogenousganglionic morphine levels stimulated by codeine exposure (T-test,P<0.001). FIG. 20C is a graph plotting the level of morphine detected inganglia from untreated animals or animals treated with norlaudanosoline(THP; 10⁻⁶ M), reticuline (10⁻⁶ M), or DA (10⁻⁶ M) alone or incombination with quinidine (10⁻⁶ M). Quinidine blocked the increase inendogenous ganglionic morphine levels stimulated by norlaudanosoline,reticuline, or DA exposure (T-test, P<0.001).

FIG. 21 is a sequence alignment of a partial sequence of nucleic acidamplified from Mytilus tissue (bottom strand; SEQ ID NO:1) aligned withnucleotide position 843 to position 1107 of the sequence set forth inGenBank accession number M20403 (top strand; SEQ ID NO:2). The boldletters represent mismatches, n's, and gaps.

FIG. 22 is a graph plotting the amount of morphine in Mytilus edulispedal ganglia following injection of tyrosine (T; 10⁻⁵ M) or tyramine(Ty; 10⁻⁵ M) into the foot of healthy, untreated animals or healthyanimals having had their pedal ganglia exposed to AMPT (10⁻⁴ M) orquinidine (10⁻⁴ M) 15 minutes post injection.

FIG. 23 is a graph plotting the percent of LPS-activated cells fromanimals pre-treated once with or without the indicated amount ofmorphine.

FIG. 24 is a graph plotting the percent of LPS-activated cells fromanimals pre-treated daily for 4 days with or without the indicatedamount of morphine.

FIG. 25 is a graph plotting the death rate of TNF-α-treated animalspre-treated daily for 4 days with or without morphine (10⁻⁷ M).

FIG. 26 is a graph plotting the median of channel fluorescence of bloodsamples pre-incubated with 50 nM (solid line) or 50 μM (dashed line) ofmorphine for the indicated times prior to LPS stimulation. Statisticalanalysis revealed a significant effect of morphine on NF-κB nuclearbinding at any time interval when compared with LPS stimulation alone(O-min morphine).

FIG. 27 is a graph plotting mu3 opiate receptor activity per gram ofmembrane protein (Bmax pg/g of membrane tissue) on the indicated day foranimals treated with saline (control), 1 μM morphine, or 0.01 μMmorphine.

FIG. 28 is a graph plotting nitric oxide release from pedal ganglia onthe indicated day for animals treated with saline (control), 10⁻⁶ Mmorphine, or 10⁻⁸ M morphine.

FIG. 29 is a graph plotting nitric oxide release from SH-SY5Y cellstreated with 10⁻⁶ M morphine (grey bar), 10⁻⁶ M morphine (black bar), or10⁻⁸ M morphine (white bar).

FIG. 30 contains graphs plotting nitric oxide release from untreatedSH-SY5Y cells challenged with 10⁻⁶ M morphine (grey bar) prior tomeasuring nitric oxide release or from SH-SY5Y cells treated with 10⁻⁶ Mmorphine (black bar) or 10⁻⁸ M morphine (white bar). The results in thetop, middle, and bottom panels were for cells treated as indicated forone, two, or seven days, respectively.

FIG. 31 is a graph plotting the relative mu3 opiate receptor geneexpression in mononuclear cells (MN) and polymorphonuclear cells (PMN)treated with 10⁻⁷M morphine-6-glucuronide alone or 10⁻⁷Mmorphine-6-glucuronide and 10⁻⁶ M CTOP.

FIG. 32 is a graph plotting band intensities for BACE-1 gene expressionin HTB-11 neuroblastoma cells. Lane 1: untreated cells; lane 2: 24-hourtreatment with 1 μM morphine; lane 3: 24-hour treatment with 5 μMmorphine.

FIG. 33 is a graph plotting band intensities for BACE-2 gene expressionin HTB-11 neuroblastoma cells treated as follows for 24 hours. Lane 1:untreated; lane 2: 1 μM morphine; lanes 3 and 4: 10 μM and 25 μM Aβ₁₋₄₂;lanes 5 and 6: 1 μM morphine with 10 μM and 25 μM Aβ₁₋₄₂.

FIG. 34 (top) is a graph plotting band intensities for BACE-1 and BACE-2gene expression in HTB-11 neuroblastoma cells treated as follows for 24hours. Lanes 1 and 4: untreated; lane 2 and 5: 1 μM morphine; lane 3 and6: 1 μM morphine pre-treated with 10 μM naloxone for twenty minutes.Lanes 1-3 contain products amplified with primers specific for BACE-1,while lanes 4-6 contain products amplified with primers specific forBACE-2. FIG. 34 (bottom) is a graph plotting BACE expression levelsstandardized against cyclophilin expression.

FIG. 35 (top) is a graph plotting band intensities for BACE-1 and BACE-2gene expression in HTB-11 neuroblastoma cells treated as follows for 24hours. Lanes 1 and 4: untreated; lane 2 and 5: 1 μM morphine; lane 3 and6: 1 μM morphine pre-treated with 10 μM L-NAME for twenty minutes. Lanes1-3 contain products amplified with primers specific for BACE-1, whilelanes 4-6 contain products amplified with primers specific for BACE-2.FIG. 35 (bottom) is a graph plotting BACE expression levels standardizedagainst cyclophilin expression.

FIG. 36 is a graph plotting band intensities for BACE-1 gene expressionin HTB-11 neuroblastoma cells treated as follows for 4 hours. Lane 1:untreated; lane 2, 3, and 4: 1 μM, 5 μM, and 10 μM SNAP; lane 5: 25 μMAβ₁₋₄₂ with 1 μM SNAP; lane 6: 25 μM Aβ₁₋₄₂ with 10 μM SNAP.

FIG. 37 is a graph plotting band intensities for BACE-1 gene expressionin HTB-11 neuroblastoma cells treated as follows for 24 hours. Lane 1:untreated; lane 2, 3, and 4: 1 μM, 5 μM, and 10 μM SNAP; lane 5: 25 μMAβ₁₋₄₂ with 1 μM SNAP; lane 6: 25 μM Aβ₁₋₄₂ with 10 μM SNAP.

FIG. 38 is a graph plotting band intensities for BACE-2 gene expressionin HTB-11 neuroblastoma cells treated as follows for 4 hours. Lane 1:untreated; lane 2, 3, and 4: 1 μM, 5 μM, and 10 μM SNAP; lane 5: 25 μMAβ₁₋₄₂ with 1 μM SNAP; lane 6: 25 μM Aβ₁₋₄₂ with 10 μM SNAP.

FIG. 39 is a graph plotting band intensities for BACE-2 gene expressionin HTB-11 neuroblastoma cells treated as follows for 24 hours. Lane 1:untreated; lane 2, 3, and 4: 1 μM, 5 μM, and 10 μM SNAP; lane 5: 25 μMAβ₁₋₄₂ with 1 μM SNAP; lane 6: 25 μM Aβ₁₋₄₂ with 10 μM SNAP.

FIG. 40 is a graph plotting band intensities for BACE-1 and BACE-2 geneexpression in HTB-11 neuroblastoma cells treated as follows for 2 hours.Lanes 1 and 4: untreated; lane 2 and 5: 1 μM SNAP; lane 3 and 6: 5 μMSNAP. Lanes 1-3 contain products amplified with primers specific forBACE-1, while lanes 4-6 contain products amplified with primers specificfor BACE-2. FIG. 40 (bottom) is a graph plotting BACE expression levelsstandardized against β-actin expression.

FIG. 41 contains graphs plotting real-time NO release from SH-SY5Yneuroblastoma cells pre-treated for 1 hour with the following amounts ofAβ₁₋₄₂ and then stimulated using 1 μM M6G at t=0. Panel A: control, 0Aβ₁₋₄₂; Panel B: 1μ Aβ₁₋₄₂; Panel C: 5 μM Aβ₁₋₄₂; Panel D: 10 μM Aβ₁₋₄₂;Panel E: 15 μM Aβ₁₋₄₂; Panel F: 25 mM Aβ₁₋₄₂; Panel G: control withL-NAME added 4 minutes before M6G.

FIG. 42 is a diagram of the possible role of NO in Alzheimer's disease.

FIG. 43A is a graph plotting NO release from human neuroblastoma cellstreated with morphine sulfate (5 μM) versus time. FIG. 43B is a bargraph plotting the peak NO release for human neuroblastoma cells treatedwith morphine sulfate (5 μM) or PBS. The control is the peak value of NOrelease from cells prior to adding morphine (i.e., basal NO release).The peak value is 22.3 nM±0.85 (p<0.001 when compared to control).

FIG. 44A is a graph plotting percent cell viability for cells receivingthe indicated treatment and either 0, 15, 30, 40, 50, or 70 μM ofrotenone for 48 hours. FIG. 44B is a bar graph plotting cell viabilityfor cells treated as indicated. FIG. 44C is a graph plotting the averageform factor measurement for cells treated as indicated. Photographs 1-6of FIG. 44 are pictures of cells corresponding to the treatmentsindicated in FIG. 44C.

FIG. 45A is a graph plotting NR1 expression levels, while FIG. 45B is agraph plotting NR2B expression levels for the following treatments:treatment #1: control; treatment #2: morphine (5 μM); treatment #3:rotenone (30 nM, LD₅₀); treatment #4: rotenone (40 nM); treatment #5:morphine+rotenone (30 nM); and treatment #6: morphine+rotenone (40 nM).Rotenone treatment caused a dose dependent decrease in NR1 expression(p<0.003, both 3 and 4 compared to 1). Morphine increased NR1 expressionat LD₅₀ (p<0.035, 5 compared to 3). Rotenone caused a dose dependentincrease in NR2B expression (p<0.001, 3 compared to 1). Morphinedecreased NR2B expression and counteracted the effects of rotenone in adose dependent manner (p<0.042, 5 compared to 3, and p<0.018, 6 comparedto 4, respectively).

FIG. 46 is a graph plotting proteasomal catalytic X subunit expressionlevels in cells treated as indicated for 4 or 24 hours. Morphineincreased the level of expression of the X subunit in a dose dependentmanner. The 5 nM morphine treatment in the presence of rotononesignificantly decreases the expression of the X subunit and wassignificant when compared to rotenone alone, p<0.014 at 4 hours, andp<0.009 at 24 hours. Rotenone also increased the level of X subunitexpression (p<0.006 at 4 hours) and (p<0.033 at 24 hours).Neuroprotection was observed with the dose dependent decrease in thelevel of proteasomal catalytic X subunit expression being significant at5 nM of morphine when compared to treatments with rotenone alone (p<0.01at 4 hours and p<0.012 at 24 hours). The values obtained with 5 μM ofmorphine plus 30 nM retenone were not statistically different fromcontrol values.

FIG. 47 is a graph plotting mRNA expression of the LMP7 immunoproteasomesubunit in cells treated as indicated for 24 hours. Although rotenonedid not cause significant increase in expression (p<0.068), there was asignificant, dose dependent decrease in LMP7 expression with morphineadministration when rotenone values were compared to morphine+rotenonevalues: p<0.026 with 1 μM morphine and p<0.018 with 5 μM morphine.

FIG. 48 contains photographs of Western blots and graphs plotting theexpression levels of 20S proteasome X subunit polypeptides in cellstreated as indicated. Morphine induced a significant dose dependentincrease in expression of X subunit after 24 hours of treatment, p<0.01(1 μM morphine compared to control) and p<0.001 (5 μM morphine comparedto control). Significant neuroprotection was observed with 5 μM morphinewhen compared to rotenone control (p<0.028). Furthermore, thistreatment, 5 μM morphine+30 nM rotenone, was not statistically differentfrom the control (p<0.065).

FIG. 49A is a graph plotting 26s chymotrypsin activity, while FIG. 49Bis a graph plotting 20S proteasome activity. Cells were treated with orwithout morphine (5 μM), rotenone (30 nM), naloxone (10 μM), and L-NAME(10 μM). A significant decrease in chymotrypsin 26S activity was causedby rotenone (p<0.043). A significant increase in chymotrypsin activitywas caused by morphine (5 μM) (p<0.021). Concomitant treatment ofmorphine and rotenone resulted in a restoring of chymotrypsin activityto the point where it was statistically insignificant to the control.Significant increase in activity of the 20S proteasome upon exposure torotenone was observed (p<0.046). Coupled with the morphine induceddecrease in 20S proteasomal function (p<0.050), there was a significantdecrease in 20S activity (p<0.034).

FIG. 50 is a graph plotting the level of free ubiquitin in cells treatedas indicated using Western blot analysis. A significant dose dependentincrease in the level of free ubiquitin was observed with morphinetreatment (p<0.001 with 5 μM morphine). A decrease was observed in thelevel of ubiquitin with rotenone treatment (p<0.064), and this wasreversed with the administration of morphine (p<0.039 when compared torotenone treatment).

FIG. 51 is a graph plotting the level of LMP7 mRNA expression in cellstreated as indicated. IFNγ (20 ng/mL) caused in increase in expressionof the LMP7 immunoproteasome subunit (p<0.001). Concomitant morphineadministration with IFNγ produced significant neuroprotection (p<0.034).

FIG. 52 is a graph plotting the level of LMP7 polypeptide expression incells treated as indicated. IFNγ caused an increase in LMP7 polypeptideexpression (p<0.001). Morphine was able to counteract this effect inconcomitant treatment with IFNγ (p<0.001 compared to IFNγ at both 36hours and 48 hours).

DETAILED DESCRIPTION

This document provides methods and materials related to using morphine,morphine precursors (e.g., tyrosine, tyramine, phenyl alanine, 3,4dihydroxyphenyl pyruvate, dihydroxyphenyl acetaldehyde, dopamine,L-DOPA, reticuline, norlaudanosoline, salutaridine, thebaine, orcodeine), morphine-6β-glucuronide, inhibitors of morphine synthesis oractivity, and inhibitors of dopamine synthesis to treat diseases, toreduce inflammation, or to restore normal function. For example, thisdocument provides compositions containing morphine, morphine precursors,morphine-6β-glucuronide, inhibitors of morphine synthesis, inhibitors ofmorphine activity, inhibitors of dopamine synthesis, or combinationsthereof. This document also provides methods for using suchcompositions.

This document provides compositions containing morphine, morphineprecursors, morphine-6β-glucuronide, or combinations thereof. Morphineor morphine-6β-glucuronide can be formulated into compositions designedto deliver a low dose of morphine or morphine-6β-glucuronide to amammal. Typically, a low dose of morphine is a dose that is below thatwhich is given to relieve a mammal of pain. For example, a low dose ofmorphine can be between 0.5 and 10 μg (e.g., between 1 and 9 μg, between1 and 8 μg, between 1 and 7 μg, between 1 and 6 μg, between 1 and 5 μg,between 2 and 10 μg, between 3 and 10 μg, between 4 and 10 μg, orbetween 5 and 10 μg) per kg of body weight per day. A low level ofmorphine-6β-glucuronide can be similar to those of morphine. Forexample, a low dose of morphine-6β-glucuronide can be between 1 and 10μg (e.g., between 1 and 9 μg, between 1 and 8 μg, between 1 and 7 μg,between 1 and 6 μg, between 1 and 5 μg, between 2 and 10 μg, between 3and 10 μg, between 4 and 10 μg, or between 5 and 10 μg) per kg of bodyweight per day. In some cases, morphine or morphine-6β-glucuronide canbe formulated to deliver between 35 and 700 μg of morphine ormorphine-6β-glucuronide for a 70 kg individual. In some cases, a lowdose can be any amount that is high enough to cause cells within themammal to release nitric oxide yet low enough to not cause the mammal toexperience analgesia. Such a dose can be, without limitation, about 5 μgper kg of body weight per day.

When given orally, morphine or morphine-6β-glucuronide can be formulatedinto a pill or tablet that contains between 10 and 1000 μg (e.g.,between 10 and 900 μg, between 10 and 800 μg, between 10 and 700 μg,between 10 and 600 μg, between 10 and 500 μg, between 30 and 1000 μg,between 35 and 1000 μg, between 40 and 1000 μg, between 50 and 1000 μg,between 35 to 700 μg, or between 35 and 500 μg) of morphine ormorphine-6β-glucuronide. For example, a tablet can be designed tocontain 100 μg of morphine. In these cases, a mammal weighing about 70kg can be instructed to take between one and three pills or tablets perday. Mammals weighing more or less than 70 kg can be instructed to takethe appropriate number of pills or tablets to achieve a similar finalconcentration. The term “morphine” as used herein includesdihydromorphine, morphine sulfate, morphine hydrochloride, and morphineacetate.

The compositions provided herein can contain one or more than one (e.g.,two, three, four, five, or more) morphine precursors without containingmorphine or morphine-6β-glucuronide. Examples of morphine precursorsinclude, without limitation, tyrosine, tyramine, dopamine, L-DOPA, 3,4dihydroxyphenyl pyruvate, dihydroxyphenyl acetaldehyde, phenylalanine,reticuline, norlaudanosoline, salutaridine, thebaine, and codeine. Asdescribed herein, a composition can be designed to contain tyrosine,tyramine, dopamine, L-DOPA, 3,4 dihydroxyphenyl pyruvate,dihydroxyphenyl acetaldehyde, phenylalanine, reticuline,norlaudanosoline, salutaridine, thebaine, codeine, or combinationsthereof. Such compositions can contain any amount of the morphineprecursors such as an amount between 1 and 10 mg per person weighingabout 70 kg. For example, a composition can contain between 1 and 10 mgof reticuline.

The compositions provided herein can contain one or more (e.g., two,three, four, five, or more) morphine precursors in addition to morphineor morphine-6β-glucuronide or in addition to a combination of morphineand morphine-6β-glucuronide. In some cases, a composition can containmorphine and reticuline. Compositions containing morphine and a morphineprecursor as well as compositions containing morphine-6β-glucuronide anda morphine precursor can contain any amount of the morphine precursorsuch as between 0.1 and 100 mg (e.g., between 0.1 and 90 mg, between 0.1and 75 mg, between 0.1 and 50 mg, between 0.1 and 25 mg, between 0.1 and10 mg, between 0.5 and 100 mg, between 1 and 100 mg, between 1 and 50mg, or between 1 and 10 mg) of the morphine precursor. For example, acomposition can contain between 10 and 100 μg of morphine, between 10and 100 μg of morphine-6β-glucuronide, and between 1 and 10 mg ofreticuline.

A composition (e.g., pill or tablet) designed to deliver a low dose ofmorphine, designed to deliver a low dose of morphine-6β-glucuronide,designed to contain one or more morphine precursors, or designed tocontain any combination thereof (e.g., both morphine and one or moremorphine precursors) can be formulated to contain additional componentssuch as L-arginine, selenium, and Ca⁺⁺. L-arginine can be included topromote a cell's ability to release nitric oxide in response to morphinevia nitric oxide synthesis from L-arginine metabolism. Selenium can beadded to enhance mu3 opiate receptor gene expression. Calcium sourcessuch as calcium citrate or CaCO₃ can be added to help facilitate themetabolism of L-arginine into nitric oxide via a calcium-dependentconstitutive nitric oxide synthase. To reduce acid reflux problems inoral applications, CaCO₃ can be used as a calcium source. In some cases,a pill or tablet designed to deliver a low dose of morphine can beformulated to contain 35 to 700 μg morphine (e.g., 0.1 mg morphine), 1mg to 500 mg L-arginine (e.g., 300 mg L-arginine), 55 μg to 200 μgselenium (e.g., 100 μg selenium), and 1000 to 1300 mg Ca⁺⁺ (e.g., 1000mg Ca⁺⁺). In some cases, a pill or tablet can be formulated to contain 1to 10 mg reticuline (e.g., 5 mg reticuline), 1 mg to 500 mg L-arginine(e.g., 300 mg L-arginine), 55 μg to 200 μg selenium (e.g., 100 μgselenium), and 1000 to 1300 mg Ca⁺⁺ (e.g., 1000 mg Ca⁺⁺). Othercomponents that can be included in a composition provided hereininclude, without limitation, pharmaceutically acceptable aqueousvehicles, pharmaceutically acceptable solid vehicles, steroids,antibacterial agents, anti-inflammatory agents, immunosuppressants,dilators, vaso-constrictors, anti-cholinergics, anti-histamines,antioxidant, and combinations thereof.

In some cases, a composition (e.g., pill or tablet) designed to delivera low dose of morphine, designed to deliver a low dose ofmorphine-6β-glucuronide, designed to contain one or more morphineprecursors, or designed to contain any combination thereof (e.g., bothmorphine and one or more morphine precursors) can be formulated tocontain one or more inhibitors of morphine synthesis (e.g., a CYP2D6 orCYP2D7 inhibitor) or activity (e.g., naloxone), one or more inhibitorsof dopamine synthesis or activity, or combinations thereof. Examples ofCYP2D6 inhibitors include, without limitation, amiodarone, chloroquine,cimetidine, clomipramine, diphenhydramine, duloxetine, fluoxetine,hydroxychloroquin, paroxetine, propafenone, propoxyphene, and quinidine,terbinafine.

A pharmaceutically acceptable aqueous vehicle can be, for example, anyliquid solution that is capable of dissolving morphine or a morphineprecursor (e.g., reticuline) and is not toxic to the particularindividual receiving the composition. Examples of pharmaceuticallyacceptable aqueous vehicles include, without limitation, saline, water,and acetic acid. Typically, pharmaceutically acceptable aqueous vehiclesare sterile. A pharmaceutically acceptable solid vehicle can beformulated such that morphine or a morphine precursor is suitable fororal administration. For example, capsules or tablets can containreticuline in enteric form. The dose supplied by each capsule or tabletcan vary since an effective amount can be reached by administratingeither one or multiple capsules or tablets. Any well knownpharmaceutically acceptable material such as gelatin and cellulosederivatives can be used as a pharmaceutically acceptable solid vehicle.In addition, a pharmaceutically acceptable solid vehicle can be a solidcarrier including, without limitation, starch, sugar, or bentonite.Further, a tablet or pill formulation of morphine or a morphineprecursor can follow conventional procedures that employ solid carriers,lubricants, and the like.

Steroids can be any compound containing a hydrocyclopentanophenanthrenering structure. Examples of steroids include, without limitation,prednisone, dexamethasone, and hydrocortisone. An antibacterial agentcan be any compound that is active against bacteria, such as penicillin,erythromycin, neomycin, gentamicin, and clindamycin. Ananti-inflammatory agent can be any compound that counteractsinflammation, such as ibuprofen and salicylic acid. An immunosuppressantcan be any compound that suppresses or interferes with normal immunefunction, such as cyclosporine. A dilator can be any compound thatcauses the expansion of an orifice, such as albuterol. Avaso-constrictor can be any compound that constricts or narrows bloodvessels, such as phenylephrine hydrochloride, cocaine, and epinephrine.An anti-cholinergic can be any compound that blocks parasympatheticnerve impulses, such as ipratropium bromide. An anti-histamine can beany compound that opposes the action of histamine or its release fromcells (e.g., mast cells), such as terfenadine and astemizole.

Any method can be used to obtain morphine, morphine-6β-glucuronide,morphine precursors, or any additional component of a compositionprovided herein. In some cases, the components of the compositionsprovided herein can be obtained using common chemical extraction,isolation, or synthesis techniques. For example, reticuline can beobtained as described elsewhere (Brochmann-Hanssen and Nielsen,Tetrahedron Lett., 18:1271-4 (1965) and U.S. Pat. No. 3,894,027). Insome cases, the components of the compositions provided herein can beobtained from commercial vendors. For example, morphine,morphine-6β-glucuronide, codeine, norlaudanosoline, and salutaridine canbe ordered from Sigma, Inc.

Any method can be used to formulate a composition provided herein. Forexample, common formulation mixing and preparation techniques can beused to make a composition having the components described herein. Inaddition, the compositions provided herein can be in any form. Forexample, a composition provided herein can be in the form of a solid,liquid, and/or aerosol including, without limitation, powders,crystalline substances, gels, pastes, ointments, salves, creams,solutions, suspensions, partial liquids, sprays, nebulae, mists,atomized vapors, tinctures, pills, capsules, tablets, and gelcaps. Insome cases, the composition can be a dietary supplement. In someembodiments, a composition containing morphine, one or more morphineprecursors, or a combination thereof can be prepared for oraladministration by mixing the components with one or more of thefollowing: a filler, a binder, a disintegrator, a lubricant, and acoloring agent. Lactose, corn starch, sucrose, glucose, sorbitol,crystalline cellulose, silicon dioxide, or the like can be used as thefiller. Polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methylcellulose, acacia, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethyl cellulose, calcium citrate, dextrin, orpectin can be used as the binder. Magnesium stearate, talc, polyethyleneglycol, silica, or hardened plant oil can be used as the lubricant. Apharmaceutically acceptable coloring agent can be used as the coloringagent. Cocoa powder, mentha water, aromatic acid, mentha oil, borneol,or powdered cinnamon bark also can be added. In some cases, acomposition containing morphine, one or more morphine precursors, or acombination thereof can be prepared for injection by mixing thecomponents with one or more of the following: a pH adjusting agent, abuffer, a stabilizer, and a solubilizing agent.

The compositions provided herein can be administered to any mammal(e.g., rat, mouse, dog, cat, horse, cow, goat, pig, monkey, or human).In addition, any route of administration (e.g., oral or parenteraladministration) can be used to administer a composition provided hereinto a mammal. For example, a composition containing morphine orreticuline can be administered orally or parenterally (e.g., asubcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, or intravenous injection).

While not being limited to any particular mode of action, thecompositions provided herein can be used to increase or maintain a basallevel of nitric oxide release by cells (e.g., cells expressing mu3opiate receptors). The administration of morphine precursors such asreticuline to a mammal can lead to the conversion of the morphineprecursor into morphine. The morphine produced from the morphineprecursor or the morphine provided directly by a composition containingmorphine or the morphine-6β-glucuronide provided directly by acomposition containing morphine-6β-glucuronide can activate mu3 opiatereceptors, which are coupled to nitric oxide release, and can downregulate the activated state of tissues within the mammal making themless excitable. For example, administering morphine or reticuline canlimit undesired excitation and restore basal activity levels within amammal. In addition, certain mammals may not produce enough endogenousmorphine to fulfill the needs of processes normally using this materialto down regulate their excitatory state (e.g., a run-awaypro-inflammatory state, mental disorders, vascular disorders).Administering a morphine precursor such as reticuline can providemammals with the morphine needed to down regulate excitatory stateswithout administering a controlled substance. Administering morphine ormorphine-6β-glucuronide directly at a low dose can provide mammals withthe morphine needed to down regulate excitatory states withouttriggering tolerance to the administered morphine ormorphine-6β-glucuronide. For example, as described herein, morphine canbe administered chronically (e.g., a long duration) at a low dosewithout observing a reduction of morphine's effects (e.g., nitric oxiderelease) over time. In addition, administering morphine-6β-glucuronidecan provide mammals with nitric oxide release in the periphery asopposed to the brain since morphine-6β-glucuronide exhibits a limitedability to cross the blood brain barrier.

The compositions provided herein can be administered to a mammal in anyamount, at any frequency, and for any duration. Typically, a compositionprovided herein can be administered to a mammal in an amount, at afrequency, and for a duration effective to induce nitric oxide releasein the mammal. In some cases, a composition provided herein can beadministered to a mammal in an amount, at a frequency, and for aduration effective to reduce the severity of a symptom of a disease orcondition (e.g., schizophrenia, mania, depression, psychosis, chronicpain, paranoia, autism, stress, Alzheimer's disease, Parkinson'sdisease, pro-inflammatory diseases, autoimmune disorders, histolyticmedullary reticulosis, lupus, arthritis, atherosclerosis, neuronalvasculopathy, or addiction).

An effective amount of a composition provided herein or of morphine orof a morphine precursor (e.g., reticuline) can be any amount thatinduces cells to release nitric oxide without producing significanttoxicity to the mammal. In some cases, an effective amount of acomposition provided herein or of morphine or of a morphine precursor(e.g., reticuline) can be any amount that reduces, prevents, oreliminates a symptom of a disease or condition upon administration to amammal without producing significant toxicity to the mammal. In somecases involving morphine precursors, an effective amount can be anyamount that results in the production of detectable amounts of morphinewithin a tissue sample.

Again, a composition provided herein can be administered to a mammal inany amount. In some embodiments, the amount of a composition providedherein or of morphine or of a morphine precursor (e.g., reticuline) canbe greater than 0.01 mg/kg of body weight. In some cases, the amount ofa composition provided herein or of morphine or of a morphine precursor(e.g., reticuline) can be between about 0.01 and about 50 mg/kg (e.g.,between about 0.01 and about 45 mg/kg; between about 0.1 and about 25mg/kg; or between about 1 and about 5 mg/kg) of body weight. Theeffective amount can vary depending upon the disease to be treated (ifany), the site of administration, and the mammal to be treated. Sucheffective amounts can be determined using the methods and materialsprovided herein. For example, the level of morphine production can beassessed using routine experimentation in vitro or in vivo. For example,a patient having a particular condition can receive 5 mg/kg body weightof reticuline. If the patient fails to respond or produce morphine, thenthe amount can be increased by, for example, ten fold. After receivingthis higher concentration, the patient can be monitored for bothresponsiveness to the treatment and toxicity symptoms, and adjustmentsmade accordingly.

Various factors can influence the actual amount used for a particularapplication. For example, the frequency of administration, duration oftreatment, combination of other agents, site of administration, stage ofdisease (if present), and the anatomical configuration of the treatedarea may require an increase or decrease in the actual amountadministered.

The frequency of administration of a composition provided herein can beany frequency. For example, the frequency of administration can be fromabout four times a day to about once a month, or more specifically, fromabout twice a day to about once a week. In addition, the frequency ofadministration can remain constant or can be variable during theduration of treatment. As with the amount administered, various factorscan influence the actual frequency of administration used for aparticular application. For example, the amount, duration of treatment,combination of agents, site of administration, stage of disease (ifpresent), and the anatomical configuration of the treated area mayrequire an increase or decrease in administration frequency. In oneembodiment, a composition containing reticuline can be administereddaily at a dose of about 1 to about 5 mg of reticuline per kg of bodyweight.

The duration of administration of a composition provided herein can beany duration. For example, a duration of administration of a compositionprovided herein can be longer than a week, month, three months, sixmonths, nine months, a year, two years, or three years. In some cases,an effective duration can be any duration that reduces, prevents, oreliminates a symptom of a disease upon administration to a mammalwithout producing significant toxicity to the mammal. Such an effectiveduration can vary from several days to several weeks, months, or years.In general, an effective duration for the treatment of an acute diseasecan range in duration from several days to several months. Onceadministration of the composition is stopped, however, disease symptomsmay return. In such cases, an effective duration for the prevention ofcertain conditions can last for as long as the individual is alive.

Multiple factors can influence the actual duration used for a particulartreatment or prevention regimen. For example, an effective duration canvary with the frequency of administration, the amount administered,combination of multiple agents, site of administration, state of disease(if present), and anatomical configuration of the treated area.

If the administration of a composition provided herein (e.g., acomposition containing reticuline) is toxic, the mammal can be treatedwith a combination of L-DOPA and dopamine to inhibit the production ofmorphine that results from the administered composition. For example, acombination of L-DOPA and dopamine can be used to reduce that amount ofmorphine produced from a composition containing a morphine precursorsuch that only 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less percentof the morphine normally produced following administration of thecomposition is actually produced.

This document also provides methods for inducing nitric oxide releasefrom cells. Such cells can be in vitro or in vivo. In addition, thecells can be any type of cell including, without limitation, neuronal,vascular, respiratory, immune, or digestive cells. To induce nitricoxide release from cells, the compositions provided herein can beadministered as described herein. For example, a composition containingmorphine can be administered to a mammal in an amount and at a frequencysuch that the mammal receives between 0.5 μg and 10 μg of morphine perkg of body weight per day for a duration of more than one month (e.g.,more than two, three, four, five, six, seven, eight, nine, or moremonths).

In addition, this document provides methods for treating a mammal havinga disease or condition using a composition provided herein. Examples ofdiseases or conditions that can be treated using the compositionsprovided herein include, without limitation, rheumatoid arthritis,systemic lupus erythematosus, systemic scleroderma, Behcet disease,periarteritis, ulcerative colitis, Crohn's disease, active chronichepatitis, glomerular nephritis, autoimmune diseases, osteoarthritis,gout, atherosclerosis, psoriasis, atopic dermatitis, pulmonary diseaseswith granuloma, encephalitis, endotoxin shock, sepsis, inflammatorycolitis, diabetes, acute myelocytic leukemia, pneumonia, hearttransplantation, encephalomylitis, anorexia, acute hepatitis, chronichepatitis, drug-induced hepatic injury, alcoholic hepatitis, viralhepatitis, jaundice, hepatic cirrhosis, hepatic insufficiency, atrialmyxoma, Castleman syndrome, multiple myeloma, Rennert T lymphomatosis,mesangial nephritis, renal cell carcinoma, cytomegaloviral hepatitis,cytomegaloviral retinopathy, adenoviral cold syndrome, adenoviralpharyngoconjunctival fever, adenoviral ophthalmia, AIDS,atherosclerosis, arteriosclerosis, vasculopathy associated withdiabetes, mania, depression, chronic pain, schizophrenia, psychosis, andparanoia. To treat a mammal having such a disease or condition, thecompositions provided herein can be administered as described herein.For example, a composition containing morphine can be administered to amammal in an amount and at a frequency such that the mammal receivesbetween 0.5 μg and 10 μg of morphine per kg of body weight per day for aduration of more than one month (e.g., more than two, three, four, five,six, seven, eight, nine, or more months). In some cases, thecompositions provided herein can be used to reduce the severity of asymptom of the disease or condition, or to prevent the development oronset of the disease or condition.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Reticuline Exposure to Invertebrate Ganglia IncreasesEndogenous Morphine Levels

The following experiments were performed to determine if exposingtissues to an opiate alkaloid precursor, reticuline, would result inincreasing endogenous morphine levels.

Material and Methods

Mytilus edulis collected from the local waters of Long Island Sound weremaintained under laboratory conditions for at least 14 days prior usingin experiments. Mussels were kept in artificial seawater (Instant Ocean,Aquarium Systems, Mentor, Ohio) at a salinity of 30 PSU and at atemperature of 18° C. as previously described (Stefano et al.,Electro-Magnetobiol., 13:123-36 (1994)).

For reticuline exposure, 400 animals were placed and maintained inartificial seawater at 24° C., whereas control animals (100) wereexposed to vehicle (PBS). For the biochemical analysis, groups of 20animals had their pedal ganglia excised at different time periods afterincubation with reticuline.

The extraction protocol, using internal or external morphine standards,was performed in a room where the animals were not maintained to avoidmorphine contamination. Single use siliconized tubes were used toprevent the loss of morphine. Mytilus edulis pedal ganglia also wereextensively washed (3 times) with PBS (0.01 M NaCl 0.132 mM, NH₄HCO₃0.132 mM; pH 7.2) prior to extraction (3 times centrifugation at 1000rpm, 1 minute, followed by discarding the PBS) to avoid exogenousmorphine contamination. Tissues were dissolved in 1N HCl and sonicatedusing a Fisher scientific sonic dismembrator 60 (Fisher Scientific,USA). The resulting homogenates were extracted with 5 mLchloroform/isopropanol 9:1.

After 5 minutes at room temperature, homogenates were centrifuged at3000 rpm for 15 minutes. The three phases were separated in thefollowing order: 1) The lowest layer corresponding to the organic phase;2) The intermediate phase corresponding to precipitated proteins; and 3)The top aqueous supernatant phase containing morphine. The supernatantwas collected and dried with a Centrivap Console (Labconco, Kansas City,Mo.). The dried extract was then dissolved in 0.05% trifluoroacetic acid(TFA) water before solid phase extraction. Samples were loaded on aWaters Sep-Pak Plus C-18 cartridge previously activated with 100%acetonitrile and washed with 0.05% TFA-water. Morphine elution wasperformed with a 10% acetonitrile solution (water/acetonitrile/TFA,89.5%:10%:0.05%, v/v/v). The eluted sample was dried with a CentrivapConsole and dissolved in water prior to high pressure liquidchromatography (HPLC) analysis.

The morphine radioimmuno-assay (RIA) determination was a solid phase,quantitative RIA, wherein ¹²⁵I-labeled morphine competes for a fixedtime with morphine in the test sample for the antibody binding site. Thecommercial kit employed was from Diagnostic Products Corporation (USA).Because the antibody was immobilized on the wall of a polypropylenetube, simply decanting the liquid phase to terminate the competition andto isolate the antibody-bound fraction of radiolabeled morphine wassufficient. The material was then counted in a Wallac, 3″, 1480 gammacounter (Perkin Elmer, USA). Comparison of the counts to a calibrationcurve yielded a measure of the morphine present in the test sample,expressed as nanograms of morphine per milliliter. The calibratorscontained, respectively, 0, 2.5, 10, 25, 75, and 250 nanograms ofmorphine per milliliter (ng/mL) in PBS. Reticuline and salutaridine didnot cross-react with the antibody. The detection limit was 0.5 ng/mL.

The HPLC analyses were performed with a Waters 626 pump (Waters,Milford, Mass.) and a C-18 Unijet microbore column (BAS). A flowsplitter (BAS) was used to provide the low volumetric flow-ratesrequired for the microbore column. The split ratio was 1/9. Operatingthe pump at 0.5 mL/minute, yielded a microbore column flow-rate of 50μL/minute. The injection volume was 5 mL. Morphine detection wasperformed with an amperometric detector LC-4C (BAS, West Lafayette,Ind.). The microbore column was coupled directly to the detector cell tominimize the dead volume. The electrochemical detection system used aglassy carbon-working electrode (3 mm) and a 0.02 Hz filter (500 mV;range 10 nA). The cell volume was reduced by a 16-μm gasket. Thechromatographic system was controlled by Waters Millennium³²Chromatography Manager V3.2 software, and the chromatograms wereintegrated with Chromatograph software (Waters).

Morphine was quantified in the tissues as described elsewhere (Zhu etal., Brain Res. Mol. Brain. Res., 88:155-60 (2001)). Briefly, the mobilephases were: Buffer A: 10 mM sodium chloride, 0.5 mM EDTA, 100 mM sodiumAcetate, pH 5.0; Buffer B: 10 mM sodium chloride, 0.5 mM EDTA, 100 mMsodium Acetate, 50% acetonitrile, pH 5.0. The injection volume was 5 mL.The running conditions were: from 0 min 0% buffer B; 10 min, 5% bufferB; at 25 min 50% buffer B; at 30 min 100% buffer B. Both buffers A and Bwere filtered through a Waters 0.22 nm filter, and the temperature ofthe whole system was maintained at 25° C. Several HPLC purificationswere performed between each sample to prevent residual morphinecontamination remaining on the column. Furthermore, mantle tissue wasrun as a negative control, demonstrating a lack of contamination (Zhu etal., Mol. Brain. Res., 117:83-90 (2003)).

For the nitric oxide assay, ten pedal ganglia (per determination)dissected from M. edulis were bathed in 1 mL sterile phosphate bufferedsaline (PBS). Experiments used morphine at a final concentration of 10⁻⁶M, naloxone at 10⁻⁶ M, and 1 μg of reticuline. For the opiate receptorantagonist experiments, ganglia were pretreated with naloxone for 10minutes prior to reticuline addition. NO release was monitored with anNO-selective microprobe manufactured by World Precision Instruments(Sarasota, Fla.). The sensor was positioned approximately 100 μm abovethe respective tissue surface. Calibration of the electrochemical sensorwas performed by use of different concentrations of a nitrosothiol donorS-nitroso-N-acetyl-DL-penicillamine (SNAP) as described elsewhere (Liuet al., Brain Res., 722:125-31 (1996)). The NO detection system wascalibrated daily. The probe was allowed to equilibrate for 10 minutes inthe incubation medium free of tissue before being transferred to vialscontaining the ganglia for another 5 minutes. Manipulation and handlingof the ganglia was only performed with glass instruments. Data wasacquired using the Apollo-4000 free radical analyzer (World PrecisionInstruments, Sarasota, Fla.). The experimental values were thentransferred to Sigma-Plot and -Stat (Jandel, CA) for graphicrepresentation and evaluation. For binding experiments, human monocytesserved as a positive control since the mu3 opiate receptor subtype,which is coupled to NO release, was identified by RT-PCR and Northernblot analysis in these cells (Cadet et al., J. Immunol., 170:5118-23(2003)). The monocytes were obtained from the Long Island Blood Center(Melville, Long Island) and processed as described elsewhere (Stefano etal., Proc. Natl. Acad. Sci., 90:11099-103 (1993); Bilfinger et al., Adv.Neuroimmunol., 3:277-88 (1993); and Magazine et al., J. Immunol.156:4845-50 (1996)).

An additional 100 excised pedal ganglia and the human monocytes wereseparately washed and homogenized in 50 volumes of 0.32 M sucrose, pH7.4, at 4° C. using a Brinkmann polytron (30 seconds, setting no. 5).The crude homogenate was centrifuged at 900×g for 10 minutes at 4° C.,and the supernatant was reserved on ice. The whitish crude pellet wasresuspended by homogenization (15 seconds, setting no. 5) in 30 volumesof 0.32 M sucrose/Tris-HCl buffer, pH 7.4, and centrifuged at 900×g for10 minutes. The extraction procedure was repeated one more time, and thecombined supernatants were centrifuged at 900×g for 10 minutes. Theresulting supernatants (S1′) were used immediately. Prior to the bindingexperiment, the S1′ supernatant was centrifuged at 30,000×g for 15minutes, and the pellet (P2) was washed once by centrifugation in 50volumes of the sucrose/Tris-HCl. The P2 pellet was then re-suspendedwith a Dounce hand-held homogenizer (10 strokes) in 100 volumes ofbuffer. Binding analysis was then performed on the cell membranesuspensions.

Aliquots of membrane suspension (0.2 mL, 0.12 mg of membrane protein)were incubated in triplicate at 22° C. for 40 minutes with theappropriate radiolabeled ligand in the presence of dextrorphan (10 mM)or levorphanol (10 mM) in 10 mM Tris-HCl buffer, pH 7.4, containing 0.1%BSA and 150 mM KCl. Free ligand was separated from membrane-boundlabeled ligand by filtration under reduced pressure through GF/B glassfiber filters (Whatman); filters were presoaked (45 minutes, 4° C.) inbuffer containing 0.5% BSA. The filters were rapidly washed with 2.5 mLaliquots of the incubation buffer (4° C.), containing 2% polyethyleneglycol 6000 (Baker). They were assayed by liquid scintillationspectrometry (Packard 460). Stereospecific binding was defined asbinding in the presence of 10 mM dextrorphan minus binding in thepresence of 10 mM levorphanol. Protein concentration was determined inmembrane suspensions (prepared in the absence of BSA).

For IC_(so) determination (defined as the concentration of drug whichelicits half-maximal inhibition of specific ³H-dihydromorphine binding(for mu3), an aliquot of the respective tissue-membrane suspension wasincubated with non-radioactive opioid compounds at 6 differentconcentrations for 10 minutes at 22° C. and then with ³H-dihydromorphinefor 60 minutes at 4° C. as described elsewhere (Stefano et al., Proc.Natl. Acad. Sci., 90:11099-103 (1993)). The mean+/−S.E.M. for threeexperiments was recorded for each compound. Tyr-D-Pen-Gly-Phe-D-Pen(DPDPE) and naltrexone were obtained from Sigma Chemical Co. (St. Louis,Mo.).

Results

Morphine was identified in the ganglionic extraction by reverse phaseHPLC using a gradient of acetonitrile following liquid and solidextraction, and was compared to an authentic standard (FIG. 1). Thematerial exhibited the same retention time as authentic morphine. Theelectrochemical detection sensitivity of morphine was 80 picograms. Theconcentration of morphine was 1.43±0.41 ng/mg±SEM ganglionic wet weightas determined by the Chromatogram Manager 3.2 commercial software(Millemmium³², Waters, Milford, Mass.) extrapolated from the peak-areacalculated for the external standard. Ganglia incubated with 50 ng ofreticuline for 1 hour exhibited a statistical increase in theirendogenous morphine levels (6.7±0.7 ng/mg tissue wet weight; P<0.01;FIG. 1).

The electrochemical results are compatible with the RIA quantification(FIG. 2), which yields a control ganglionic level of morphine at1.33±0.61 ng/mg tissue wet weight±SEM. Incubation with variousconcentrations of reticuline increases ganglionic morphine levels afterone hour in a concentration dependent manner (FIG. 2). Exposure ofexcised ganglia to 100 ng of reticuline yields about 14.53±4.6 ng/mgmorphine (FIG. 2; P<0.001). The increase in ganglionic morphine levelsoccurred gradually over the 60-minute incubation period, beginning 10minutes post reticuline addition (FIG. 3). From these studies, about 24percent of the added reticuline was converted to morphine. Blank runsbetween morphine HPLC determinations did not show a morphine residuewith RIA, nor did any signs of its presence occur with mantle tissue.Incubation of 50 ng of reticuline with mantle tissues did not producedetectable morphine.

Pedal ganglia, which contain mu opiate receptors, respond to morphineexposure by releasing constitutive nitric oxide synthase derived NO in analoxone and L-NAME sensitive manner (Stefano et al., Brain Res.,763:63-8 (1997) and Cadet et al., Mol. Brain. Res., 74:242-6 (1999)). Inan attempt to substantiate the identity of newly synthesized morphinefurther, ganglia were examined for their ability to release NO inresponse to reticuline exposure (FIG. 4; Table 1). Reticuline (10⁻⁷ M)did not stimulate ganglionic NO release in a manner resembling that ofmorphine (10⁻⁶ M), which releases NO seconds after its application tothe ganglia and lasts for 5 minutes (FIG. 4; Table 1). Instead, withreticuline, there was a three-minute delay, which was followed by anextended release period occurring over 17 minutes (FIG. 4). Thisreticuline-stimulated release occurred because reticuline was beingconverted to morphine and the newly synthesized morphine was responsiblefor the detected NO release, as indicated by the time course of morphineincrease following reticuline exposure (FIG. 3).

TABLE 1 NO release from pedal ganglia. NO Peak NO Peak LIGAND Level (nM)Time (min) Control  0.9 ± 0.1 None Morphine (10⁻⁷ M) 24.3 ± 3.1 0.8 ±0.2 Reticuline (10⁻⁷ M) 17.6 ± 3.8 9.2 ± 1.5 Salutaridine (10⁻⁷ M) 18.5± 3.3 8.9 ± 1.3 Dihydromorphine (10⁻⁷ M) 23.2 ± 3.7 0.9 ± 0.3

The reticuline and salutaridine NO peak time and latency before NOproduction rose at 10 nM were statistically different (P<0.01) fromthose values recorded for morphine and dihydromorphine.

Neither reticuline nor salutaridine exhibited binding affinity for thepedal ganglia mu3 opiate receptor subtype (Table 2). This finding wasfurther substantiated using the positive control of human monocytes fromwhich the mu3 opiate receptor subtype was cloned. This result indicatesthat the pre-treatment of the ganglia with naloxone (10⁻⁶ M) blockingthe reticuline (10⁻⁷ M) stimulated release of NO (FIG. 4) occurs by wayof this precursor being converted to morphine since reticuline does nothave an affinity for the mu3 opiate receptor (Table 2).

TABLE 2 Displacement of ³H-dihydromorphine (DHM; nmol-L⁻¹) by opioidligands in various tissue membrane suspensions. Pedal Ganglia MonocytesLIGAND (IC⁵⁰; nM) (IC⁵⁰; nM) δ-agonists DPDPE >1000 >1000 μ-agonistsReticuline >1000 >1000 Salutaridine >1000 >1000 Dihydromorphine 22 ± 2.319.1 ± 3.3 Antagonists Naltrexone 31 ± 7.1 34.6 ± 8.2 DPDPE = (D-Pen²,D-Pen⁵)-enkephalin.

The results provided herein demonstrate that (1) morphine is present inMytilus pedal ganglia; (2) exposing pedal ganglia to reticuline resultsin significant increases in ganglionic morphine levels in aconcentration and time dependent manner; (3) reticuline stimulatesganglionic NO production, following a latency period, in a mannerconsistent with it being converted to morphine; and (4) reticuline doesnot exhibit an affinity for the mu3 opiate receptor, also suggestingthat NO release occurs because of the conversion of reticuline intomorphine.

Example 2 Mammalian Cells Produce Morphine from Reticuline

Human cells (NCI-H295R) were adapted from the NCI-H295 pluripotentadrenocortical carcinoma cell line (ATCC CRL-10296), which is from acarcinoma of the adrenal cortex. The original cells were adapted to aculture medium that decreased the population doubling time from 5 daysto 2 days. While the original cells grew in suspension, the adaptedcells were selected to grow in a monolayer. These cells retained theability to produce adrenal androgens and were responsive to angiotensinII and potassium ions. To propagate these cells, the culture medium wasa 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 mediumcontaining 15 mM HEPES, 0.00625 mg/mL insulin, 0.00625 mg/mLtransferrin, 6.25 ng/mL selenium, 1.25 mg/mL bovine serum albumin, and0.00535 mg/mL linoleic acid, 97.5%; Nu-Serum I, 2.5%. See, e.g., Raineyet al., Mol. Cell. Endocrinol., 100:45-50 (1994); Gazdar et al., CancerRes., 50:5488-5496 (1990); and Bird et al., Endocrinology, 133:1555-1561(1993)).

The cells were subcultured in 6 well cell culture cluster (Corning Inc.)24 hours before the experiment. The amount of cells was determined byMicroscope (Nickon inc). Various amounts of reticuline were added to thecells, and the cells were cultured with an NAPCO incubator. Theincubation was terminated after 24 hours by adding 10 NHCl. Morphine inthe cells and culture medium was detected with an RIA kit purchased fromDiagnostic Products Cooperation, CA, USA.

The morphine levels in the cells were significantly higher whenincubated with reticuline. One half million cells incubated with 100 ngof reticuline produced about 28±5.4 ng of morphine. Control cells onlyproduced about 9.6±3.5 ng of morphine. Culture media were negative inthe test. These results demonstrate that human cells can producemorphine from reticuline.

Example 3 The Combination of L-DOPA and Dopamine Inhibits EndogenousMorphine Production

Mytilus pedal ganglia were obtained and incubated with 10 μg L-DOPAalone, 10 μg dopamine alone, or 10 μg L-DOPA plus 10 μg dopamine. Thecontrol ganglia were not incubated with L-DOPA or dopamine and exhibited11.9 ng/mL morphine. The ganglia incubated with either L-DOPA alone ordopamine alone exhibited 9.31 and 8.82 ng/mL morphine, respectively. Incontrast, ganglia incubated with both L-DOPA and dopamine exhibited 5.52ng/mL of morphine. These results demonstrate that treatment with bothL-DOPA and dopamine can reduce morphine production.

Example 4 L-DOPA Increase Production of Morphine

M. edulis collected from the local waters of Long Island Sound weremaintained at a salinity of 30 PSU and at a temperature of 18° C. inmarine aquaria as described elsewhere (Stefano et al.,Electro-Magnetobiol., 13:123-36 (1994)). For in vitro ganglionic assays,groups of 10 animals had their pedal ganglia excised and examined fortheir morphine levels at different time periods following the additionof L-DOPA or reticuline and at different concentrations of thesemorphine precursors. In vitro incubation with reticuline served as apositive control since the results provided in Example 1 demonstratethat reticuline increases endogenous ganglionic morphine levels. L-DOPAwas incubated with ganglia at concentrations ranging from 1 to 100 ng/mLand at different times.

For in vivo precursor injection experiments, the animal's foot wasinjected with either reticuline or L-DOPA (0.1 and 1 μg/injection,respectively). Chemicals were injected by BD 1 cc syringes with 26 Gneedles. Each needle was inserted into that base of the foot just abovethe pedal ganglia.

Morphine Determination, Solid Phase Extraction

The morphine extraction protocol was performed using dissected andpooled ganglia, obtained from in vitro and in vivo experiments and runseparately, as described herein.

The dried extract was then dissolved in 0.05% trifluoroacetic acid (TFA)water before solid phase extraction. Samples were loaded on a WatersSep-Pak Plus C-18 cartridge previously activated with 100% acetonitrileand washed with 0.05% TFA-water. Morphine elution was performed with a10% acetonitrile solution (water/acetonitrile/TFA, 89.5%:10%:0.05%,v/v/v). The eluted sample was dried with a Centrivap Console anddissolved in water prior to HPLC analysis.

Radioimmuno-Assay (RIA) Determination

The morphine RIA determination was a solid phase, quantitative RIA,wherein ¹²⁵I-labeled morphine competes for a fixed time with morphine inthe test sample for the antibody binding site. The commercial kit usedwas obtained from Diagnostic Products Corporation (USA). The detectionlimit was 0.5 ng/mL.

HPLC and Electrochemical Detection of Morphine in Samples

The HPLC analyses were performed with a Waters 626 pump (Waters,Milford, Mass.) and a C-18 Unijet microbore column (BAS). A flowsplitter (BAS) was used to provide the low volumetric flow-ratesrequired for the microbore column. The split ratio was 1/9. Operatingthe pump at 0.5 mL/min, yielded a microbore column flow-rate of 50μL/min. The injection volume was 5 μL. Morphine detection was performedwith an amperometric detector LC-4C (BAS, West Lafayette, Ind.). Themicrobore column was coupled directly to the detector cell to minimizethe dead volume. The electrochemical detection system used a glassycarbon-working electrode (3 mm) and a 0.02 Hz filter (500 mV; range 10nA). The cell volume was reduced by a 16-μm gasket. The chromatographicsystem was controlled by Waters Millennium³² Chromatography Manager V3.2software, and the chromatograms were integrated with Chromatographsoftware (Waters).

Morphine was quantified in the tissues using methods described elsewhere(Zhu et al., Brain Res. Mol. Brain. Res., 88:155-60 (2001)). SeveralHPLC purifications were performed between each sample to preventresidual morphine contamination remaining on the column. Mantle tissuewas run as a negative control, demonstrating a lack of contamination.Morphine was not found in any of the solutions used in theseexperiments. Furthermore, animals injected with 5-hydroxytryptophan organglia incubated with this serotonin precursor did not exhibit anychanges in their endogenous ganglionic morphine levels.

Results

Incubation of pedal ganglia in vitro with various concentrations ofL-DOPA or reticuline increased ganglionic morphine levels in a time andconcentration dependent manner (FIGS. 5 and 6). Control ganglia, exposedto vehicle or 5-hydroxytryptophan (1 μg/gm tissue), a serotoninprecursor, exhibited 2.1±0.44 and 2.1±0.41 ng morphine per ganglion wetweight, respectively, whereas those exposed to L-DOPA exhibited 3.6±0.45ng morphine per ganglion, representing a statistically significantincrease (P<0.05). Exposure of excised ganglia to 100 ng of reticulineresulted in about 5.0±0.47 ng morphine per mg ganglion (FIGS. 5 and 6;P<0.001). The increase in ganglionic morphine levels, after L-DOPAexposure, occurs gradually over the 60 minute incubation period,beginning 10 minutes post exposure (FIG. 6). From these results, about5% of L-DOPA appears to be converted to morphine. Blank runs betweenmorphine HPLC determinations as well as running negative tissuecontrols, i.e., mantle, did not reveal a morphine residue with RIA.Analysis of the marine water and various chemicals used in the protocolalso lacked morphine.

The following was performed to determine if injection of these sameprecursors into intact healthy animals would yield an increase inmorphine levels. Injecting animals with either reticuline or L-DOPAsignificantly increased pedal ganglionic morphine levels after one hour(FIG. 7), demonstrating that morphine synthesis occurred. Injection of5-hydroxytryptaphan failed to increase ganglionic morphine levels. Theseresults demonstrate that reticuline and L-DOPA can be administered to ananimal so that the animal can produce additional morphine.

The results provided herein also demonstrate that L-DOPA can be used inboth morphinergic as well as dopaminergic pathways. About 5 percent ofL-DOPA, which occurs early in the synthesis scheme in both pathways,appears to be used for morphine synthesis, compared to about 25 percentof reticuline, which is closer to the end product morphine and thereforemore dedicated to morphine synthesis.

In addition, these results together with the results from Example 4appear to indicate that high doses of L-DOPA can inhibit morphineproduction, while low doses of L-DOPA can result in increased morphineproduction. One possible mechanism is that high doses of L-DOPA exceededan inhibitory threshold thereby leading to inhibition of morphineproduction. Low L-DOPA doses can by-pass this inhibitory threshold.

Example 5 Norlaudanosoline Increases Production of Morphine

Mytilus edulis collected from the local waters of Long Island Sound weremaintained as described elsewhere (Stefano et al., Electro-Magnetobiol.,13: 123-36 (1994)). For the biochemical analysis, groups of 20 animalshad their pedal ganglia excised at different time periods and incubatedwith different concentrations of norlaudanosoline, ranging from 1 to 100ng/mL.

Morphine Determination, Solid Phase Extraction

The morphine extraction protocol was performed in pooled ganglia asdescribed herein. The dried extract was then dissolved in 0.05%trifluoroacetic acid (TFA) water before solid phase extraction. Sampleswere loaded on a Waters Sep-Pak Plus C-18 cartridge previously activatedwith 100% acetonitrile and washed with 0.05% TFA-water. Morphine elutionwas performed with a 10% acetonitrile solution (water/acetonitrile/TFA,89.5%:10%:0.05%, v/v/v). The eluted sample was dried with a CentrivapConsole and dissolved in water prior to HPLC analysis.

Radioimmuno-Assay (RIA) Determination

The morphine RIA determination was a solid phase, quantitative RIA,wherein ¹²⁵I-labeled morphine competes for a fixed time with morphine inthe test sample for the antibody binding site. The commercial kit usedwas obtained from Diagnostic Products Corporation (USA). The detectionlimit was 0.5 ng/mL.

HPLC and Electrochemical Detection of Morphine in the Sample

The HPLC analyses were performed with a Waters 626 pump (Waters,Milford, Mass.) and a C-18 Unijet microbore column (BAS). A flowsplitter (BAS) was used to provide the low volumetric flow-ratesrequired for the microbore column. The split ratio was 1/9. Operatingthe pump at 0.5 mL/minute, yielded a microbore column flow-rate of 50μL/minute. The injection volume was 5 μL. Morphine detection wasperformed with an amperometric detector LC-4C (BAS, West Lafayette,Ind.). The microbore column was coupled directly to the detector cell tominimize the dead volume. The electrochemical detection system used aglassy carbon-working electrode (3 mm) and a 0.02 Hz filter (500 mV;range 10 nA). The cell volume was reduced by a 16-μm gasket. Thechromatographic system was controlled by Waters Millennium32Chromatography Manager V3.2 software, and the chromatograms wereintegrated with Chromatograph software (Waters).

Morphine was quantified in the tissues using methods described elsewhere(Zhu et al., Brain Res. Mol. Brain. Res., 88:155-60 (2001)). SeveralHPLC purifications were performed between each sample to preventresidual morphine contamination remaining on the column. Furthermore,mantle tissue was run as a negative control, demonstrating a lack ofcontamination.

Results

Incubation of the ganglia in vitro with various concentrations ofnorlaudanosoline (also called tetrahydropapoverine (THP)) increasedganglionic morphine levels after one hour in a concentration and timedependent manner (FIGS. 8 and 9; P<0.01). The increase in in vitroganglionic morphine levels, after norlaudanosoline exposure, occurredgradually over the 60 minute incubation period (FIG. 9). About 20percent of norlaudanosoline appears to be converted into morphine. Blankruns between morphine HPLC determinations as well as running negativetissue controls, i.e., mantle, did not reveal a morphine residue withRIA. Analysis of the marine water and various chemicals used in theprotocol also demonstrated a lack of morphine.

Example 6 Producing Morphine in Human Cells

Human peripheral blood cells were obtained from the Long Island BloodServices (Melville, N.Y.). ACK lysis buffer (8.29 g NH₄Cl, 0.15 M; 1 gKHCO₃, 1.0 mM, 37.2 mg Na₂EDTA, 0.1 mM, adding 800 mL H₂O and adjustingthe pH to 7.2-7.4 with 1 N HCl; filter sterilized through a 0.2 nmfilter and stored at room temp) was used to remove any red blood cellsfrom the buffy coat containing leukocytes. Cells were incubated for 5minutes at room temperature in lysis buffer, and RPMI media (ATCC) usedto stop the lysis reaction, followed by centrifugation for 10 minutes at200 g. The supernatant was decanted, and the pellet washed with RPMImedia. The leukocytes were resuspended in RPMI media by pipetting.

Polymorphonuclear cells (PMNs) were isolated (Ficoll-Hypaque density of1.077-1.080 g/mL) as described elsewhere (Magazine et al., J. Immunol.,156:4845 (1996); Stefano et al., Proc. Natl. Acad. Sci. USA, 89:9316(1992); and Makman et al., J. Immunol., 154:1323 (1995)), and the cellswere examined microscopically. Greater than 95 percent of the cells wereviable as determined by trypan blue exclusion.

A two-way ANOVA was used for statistical analysis after precursorexposure to the cells. Each experiment was performed 4 times. The meanvalue was combined with the mean value taken from 4 other replicates.The SEM represents the variation of the mean of the means. All drugswere purchased from Sigma Chemical CO. (St. Louis, Mo.), exceptbufuralol, which was purchased from BD Biosciences Clontech (MountainView, Calif.).

The medium containing the PMNs was then separated after and beforeprecursor exposure at varying concentrations for 1 hour. Cells werewashed, and the endogenous morphine content was determined The medium,devoid of cells, was also examined for the presence of morphine.

Morphine Determination

The morphine extraction protocol was performed upon washed and pelletedWBC and PMN after the incubation period as described elsewhere (Zhu etal., Brain Res. Mol. Brain. Res., 88:155 (2001); Zhu et al., Eur. J.Mass Spect., 7:25 (2001); Zhu et al., Neuroendocrinol. Lett., 23:329(2002); Zhu et al., Mol. Brain Res., 117:83 (2003); and Zhu and Stefano,Neuro. Endocrinol. Lett., 25:323 (2004)). The dried extract wasdissolved in 0.05% trifluoroacetic acid (TFA) water before solid phaseextraction. Samples were loaded on a Waters Sep-Pak Plus C-18 cartridgepreviously activated with 100% acetonitrile and washed with 0.05%TFA-water. Morphine elution was performed with a 10% acetonitrilesolution (water/acetonitrile/TFA, 89.5%:10%:0.05%, v/v/v). The elutedsample was dried with a Centrivap Console and dissolved in water priorto HPLC analysis.

The HPLC analyses were performed with a Waters 626 pump (Waters,Milford, Mass.) and a C-18 Unijet microbore column (BAS). A flowsplitter (BAS) was used to provide the low volumetric flow-ratesrequired for the microbore column. The split ratio was 1/9. Operatingthe pump at 0.5 mL/min yielded a microbore column flow-rate of 50μL/minute. The injection volume was 5 μL. Morphine detection wasperformed with an amperometric detector LC-4C (BAS, West Lafayette,Ind.). The microbore column was coupled directly to the detector cell tominimize the dead volume. The electrochemical detection system used aglassy carbon-working electrode (3 mm) and a 0.02 Hz filter (500 mV;range 10 nA). The cell volume was reduced by a 16-μm gasket. Thechromatographic system was controlled by Waters Millennium³²Chromatography Manager V3.2 software, and the chromatograms wereintegrated with Chromatograph software (Waters).

The level of morphine in the PMN was quantified as described elsewhere(Zhu et al., Brain Res. Mol. Brain. Res., 88:155 (2001)). Several blankHPLC purifications were performed between each sample to preventresidual morphine contamination remaining on the column. Furthermore,mantle tissue was run as a negative control, demonstrating a lack ofcontamination. All solutions, media, etc. were also examined for anypresence of morphine. The results of these tests revealed a lack ofmorphine contamination.

Radioimmuno-Assay (RIA) Determination

The morphine RIA determination was a solid phase, quantitative RIA,wherein ¹²⁵I-labeled morphine competes for a fixed time with morphine inthe test sample for the antibody binding site. The commercial kit usedwas obtained from Diagnostic Products Corporation (USA). The detectionlimit was 0.5 ng/mL.

CYP2D6 Molecular Demonstration

Human heparinized whole blood obtained from volunteer blood donors (LongIsland Blood Services; Melville, N.Y.) was immediately separated using1-Step Polymorphs (Accurate Chemical and Scientific Corporation,Westbury, N.Y.) gradient medium. Five mL of heparinized blood waslayered over 5 mL of polymorphs in a 14 mL round-bottom tube and thencentrifuged for 35 minutes at 500×g in a swinging-bucket rotor at 18° C.After centrifugation, the top band at the sample/medium interfaceconsisting of mononuclear cells (MN) and the lower band consisting ofpolymorphonuclear cells (PMN) were harvested in 14 mL tubes and thenwashed with PBS (Invitrogen, Carlsbad, Calif.) by centrifugation for 10minutes at 400×g.

Isolation of Total RNA

MN and PMN cells (5×10⁶) were pelleted by centrifugation, and total RNAwas isolated with the RNeasy Protect Mini Kit (Qiagen, Stanford,Calif.). Pelleted cells were resuspended in buffer RLT and homogenizedby passing the lysate 5 times through a 20-gauge needle fitted to asyringe. The samples were then processed following the manufacturer'sinstructions. In the final step, the RNA was eluted with 50 μL ofRNase-free water by centrifugation for 1 minute at 10,000 rpm.

Reverse Transcription-Coupled Polymerase Chain Reaction (RT-PCR)

First-strand cDNA synthesis was performed using random primers(Invitrogen, Carlsbad, Calif.). 1 μg of total RNA was denatured at 95°C. and reverse transcribed at 40° C. for 1 hour using Superscript IIIRnase H-RT (Invitrogen, Carlsbad, Calif.). Ten microliters of the RTproduct was added to the PCR mix containing specific primers for theCYP2D6 gene and Platinum Taq DNA polymerase (Invitrogen, Carlsbad,Calif.). The forward primer sequence was5′-AGGTGTGTCTCGAGGAGCCCATTTGGTA-3′ (SEQ ID NO:3) and reverse primer was5′-GCAGAAAGCCCGACTCCTCCTTCA-3′ (SEQ ID NO:4). The PCR reaction wasdenatured at 94° C. for 5 minutes followed by 40 cycles at 95° C. for 1minute, 60° C. for 1 minute, and 72° C. for 1 minute, and then anextension step cycle at 72° C. for 10 minutes. PCR products wereanalyzed on a 2% agarose gel (SIGMA, St. Louis, Mo.) stained withethidium bromide. The expected sizes of the PCR products were 700 bp,300 bp, and others as described elsewhere Zhuge and Yu, World J.Gastroenterol., 10:3356 (2004).

Computer-Assisted Cell Activity Analysis

PMNs, obtained as described herein, were also processed for imageanalysis of cell conformation as described elsewhere (Schon et al., Adv.Neuroimmunol., 1:252 (1991)). The morphological measurements of PMNswere based on cell area and perimeter determinations by the use of imageanalysis software (Compix, Mars, Pa.). Form-factor (FF) calculationswere performed as described elsewhere (Stefano et al., Proc. Natl. Acad.Sci. USA, 89:9316 (1992); Stefano et al., Proc. Natl. Acad. Sci. USA,90:11099 (1993); and Stefano et al., J. Neuroimmunol., 47:189 (1993)).The observational area used for measurement determinations andframe-grabbing was 0.4 nm in diameter. The computer-assisted imageanalysis system (Zeiss Axiophot fitted with Nomarski and phase contrastoptics) was the same as described elsewhere (Stefano et al., Proc. Natl.Acad. Sci. USA, 89:9316 (1992); Stefano et al., Proc. Natl. Acad. Sci.USA, 90:11099 (1993); and Stefano et al., J. Neuroimmunol., 47:189(1993)).

The cells were analyzed for conformational changes indicative of eitheractivation (amoeboid and mobile) or inhibition (round and stationary)((Stefano et al., Proc. Natl. Acad. Sci. USA, 89:9316 (1992); Stefano etal., Proc. Natl. Acad. Sci. USA, 90:11099 (1993); and Stefano et al., J.Neuroimmunol., 47:189 (1993)). The lower the FF number, the longer theperimeter and the more amoeboid the cellular shape. The proportion ofactivated cells was determined as described elsewhere ((Stefano et al.,Proc. Natl. Acad. Sci. USA, 89:9316 (1992); Stefano et al., Proc. Natl.Acad. Sci. USA, 90:11099 (1993); and Stefano et al., J. Neuroimmunol.,47:189 (1993)).

All pharmacological agents were purchased from Research BiochemicalsIncorporated (Natick, Mass.) or Sigma (St. Louis, Mo.).

Results

In control (vehicle exposed) white blood cells (WBC), morphine wasidentified at a level of 12.33±5.64 pg/million cells±SEM (FIG. 10).These cells were extensively washed in serum-free RPMI medium, limitingany plasma morphine that may be found on the cells. It, however, ispossible that the cells nonspecifically accumulated morphine fromplasma. In order to determine if WBC contain morphine due to endogenoussynthesis, cells were incubated with specific morphine precursors,including the amino acid tyramine Tyramine, norlaudanosoline (THP),reticuline, and L-DOPA significantly increased WBC morphineconcentrations above those found in untreated cells (ANOVA test,P<0.001; FIG. 11). Morphine concentrations in cells incubated withprecursors were 90.25±10.42 pg, 136.04±8.71 pg, and 136.5±12.43pg/million cells after a one-hour treatment with THP, reticuline, andL-DOPA, respectively. Furthermore, morphine concentrations increasedwith exposure to precursors in a concentration-dependent manner (FIG.11). These results demonstrate that WBC contain low but physiologicallysignificant quantities of morphine and that exposure of these cells tomorphine precursors can increase morphine synthesis.

To identify a specific population of WBC capable of synthesizingmorphine, PMNs were examined Morphine was found in these cells at alevel of 11.2±4.21 pg/million cells (FIG. 12). Exposing PMNs to morphineprecursors including tyramine, at levels that increased morphineproduction in WBC, resulted in a statistically significant increase inmorphine concentrations in PMNs (FIGS. 12 and 13).

To determine if CYP2D6 is involved in morphine synthesis in PMNs, PMNswere incubated with tyramine and a CYP2D6 substrate (bufuralol).Treatment with both tyramine and bufuralol resulted in significantlydiminished synthesis of morphine (P<0.001 compared to precursoraugmentation levels; FIG. 12). In addition, the CYP2D6 inhibitor,quinidine, blocked morphine synthesis when PMNs were treated withtyramine, THP, or codeine, further demonstrating that CYP2D6 is involvedin the synthesis of morphine (FIG. 13). Further, CYP2D6 was found to beexpressed in PMNs as evidenced by RT-PCR expression analysis thatresulted in an amplified 306 bp fragment corresponding to the enzyme.Sequence analysis of this fragment revealed 100 percent homology withhuman CYP2D6. These results demonstrate that CYP2D6 is expressed inhuman PMNs and that it is involved in morphine synthesis.

PMN incubation medium was evaluated to determine if morphine found inPMNs would also be found in the PMN incubation medium following exposureto morphine precursors. The levels of morphine detected in media fromPMNs (3×10⁶ cells) treated with THP (100 ng), reticuline (50 ng), L-DOPA(100 ng), or L-tyrosine (100 ng) were significantly (One-way ANOVA,P<0.05) higher than the levels detected in media from untreated cells(Table 3).

TABLE 3 Morphine levels in media after the cells were removed. ControlMedium THP Reticuline L-DOPA L-tyrosine 0.726 ± 0.13 2.028 ± 0.42 2.112± 0.33 1.234 ± 0.26 2.223 ± 0.38

To examine a possible physiologic role of PMN-derived morphine,precursor-treated PMNs were incubated with other PMNs that had beenexposed to different experimental protocols. After this incubation, thePMNs were evaluated for their activity level via computer-assisted imageanalysis. Untreated PMNs exhibited a 7.3±2.1% level of activation(FF>0.6) compared to a 43.4±5.7% level of activation for cells treatedwith IL-1β (2 ng/mL) after one hour. PMNs incubated with L-DOPA (10⁻⁶ M;10⁶ cells) exhibited a 3.7±0.4% level of activation. After washing PMNsseparately and mixing the populations (L-DOPA treated and IL-1β treated)in a 1:1 ratio, the percent of activated cells decreased to 12.5±3.7% inthe mixed PMN population (FIG. 14). In performing the same experimentbut co-treating the IL-1β-treated cells with naloxone and then mixingthem with the L-DOPA-treated PMNs, the cells exhibited a 35.2±6.3% levelof activation, indicating that morphine mediated the reduced level ofactivation since naloxone significantly blocked morphine's action.

Taken together, these results demonstrate that normal, human white bloodcells such as PMNs contain endogenous morphine, have the ability tosynthesize morphine, and can release morphine into their environment. Inaddition, cells such as PMNs exposed to morphine precursors can releasemorphine into their environment, which can influence the activity stateof the same cells as well as other cells not exposed to the precursors.These results also demonstrate that WBC express CYP2D6, an enzymecapable of synthesizing morphine from tyramine, norlaudanosoline, andcodeine. In addition, the results provided herein demonstrate thatmorphine can be synthesized by another pathway, via L-DOPA, indicatingthat the dopamine and morphine biosynthesis pathways are coupled (FIG.15). Taken together, the results provided herein demonstrate thatmorphine can be made from two starting points, and that inhibitingeither pathway separately results in continued morphine synthesisapparently because the other pathway can compensate for the inhibition.

Example 7 Tyrosine and Tyramine Increase Morphine and Dopamine Levels InVitro and In Vivo

Mytilus edulis collected from the local waters of Long Island Sound weremaintained as described herein. For the biochemical in vitro analysis ofeither dopamine (DA) or morphine, groups of 20 animals had their pedalganglia excised on ice at different time periods and incubated withdifferent concentrations of tyrosine or tyramine, ranging from 1 to 100ng/mL. Ganglia were maintained in a 50:50 mixture of boiled cell-freeartificial sea water and Instant Ocean (Boston, Mass.). The pedalganglia were incubated with and without tyrosine or tyramine in thepresence of quinidine, a CYP2D6 inhibitor, and alpha methyl paratyrosine (AMPT), which inhibits tyrosine hydroxylase.

For in vivo treatments, the animal's foot (20 animals per treatment) wasinjected with tyrosine (10⁻⁵ M), tyramine (10⁻⁵ M), or saline. Otheranimals were exposed to the enzyme inhibitors AMPT or quinidine alone orimmediately following the respective foot injection. After a 1-hourincubation in seawater at room temperature, ganglionic morphine levelswere determined via the HPLC and RIA methods described herein. Allchemicals were purchased from Sigma (St. Louis, Mo.).

Extraction and HPLC UV Detection of DA

Dopamine was extracted from both ganglia (20 pedal ganglia pertreatment; replicated 4 times) and hemolymph (10 mL per treatment;replicated 4 times). After ganglionic dissection, ganglia were pooledinto one eppendorf tube, 1 ml of 1 N HCl was added, and the tissue wassonicated by sonic dismemberator (Fisher scientific, USA). Homogenizedtissue was then transferred to a 15 mL polypropylene centrifuge tube(Fisher Scientific, PA, USA). 5 mL of chloroform/isopropanol (9:1, v/v)was added, and the contents of the tube vigorously vortexed for 5minutes at room temperature. Tubes were centrifuged at 4000 rpm for 15minutes at 4° C. Supernatant (water soluble layer) was dispatched intopre-siliconized 1.5 tubes (Midwest Scientific) and kept at 4° C. forimmediate use for HPLC determination or stored at −80° C. for furtheranalysis.

HPLC was performed with waters 626 pump and 2487 dual 2, absorbancedetector. A Xterra RP18 column with 5μ size particle was used to purifydopamine Isocratic mobile phase was applied with one buffer (1 mMCH₃COONH₄, 98% distilled water and 2% HPLC grade of acetonitrile (FisherScientific). Follow rate was set at 0.5 mL/minute. A concentration curvewas obtained by running different contractions of dopamine. Thedetection limit was 0.5 μg/mL.

CYP2D6 Molecular Demonstration and Isolation of Total RNA

Pedal ganglia (100) were immediately processed after dissection. Theganglia were placed in 1.5 mL tubes and then washed with PBS(Invitrogen, Carlsbad, Calif.). Total RNA was isolated using the RNeasymini kit (Qiagen, Valencia, Calif.). Ganglia were homogenized in 600 μLbuffer RLT. The samples were processed following the manufacturer'sinstructions. In the final step, the RNA was eluted with 50 μL ofRNase-free water by centrifugation for 1 minute at 10,000 rpm.

Reverse Transcription-Coupled Polymerase Chain Reaction (RT-PCR)

First-strand cDNA synthesis was performed using random primers(Invitrogen, Carlsbad, Calif.). 1 μg of total RNA was denatured at 95°C. and reverse transcribed at 40° C. for 1 hour using Superscript IIIRnase H-RT (Invitrogen, Carlsbad, Calif.). Ten microliters of the RTproduct was added to the PCR mix containing primers for CYP2D6 andCYP2D7 genes and Platinum Taq DNA polymerase (Invitrogen, Carlsbad,Calif.). The forward primer sequence was 5′-GGCCAAGGGGAACCCTGAGA-3′ (SEQID NO:5) and reverse primer was 5′-GGTCATACCCAGGGGGACGA-3′ (SEQ IDNO:6). The PCR reaction was denatured at 95° C. for 5 minutes followedby 40 cycles at 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C.for 1 minute, and then an extension step cycle at 72° C. for 10 minutes.PCR products were analyzed on a 2% agarose gel (Sigma, St. Louis, Mo.)stained with ethidium bromide. The expected sizes of the PCR productswere 282 bp for CYP2D6 and 340 bp for CYP2D7.

DNA Sequencing

After excising the PCR product from the gel, DNA purification wasperformed with the Qiaquick gel extraction kit (Qiagen). The PCR productwas dissolved in 35 μL H₂O and sent to Lark Technologies, Inc. (Houston,Tex.) for direct sequencing.

Results

Mytilus pedal ganglia were incubated in vitro with tyrosine or tyramine,both of which resulted in an increase in ganglionic morphine levels in aconcentration and time dependent manner (FIG. 16; P<0.001, from1.08±0.27 ng/g ganglionic wet weight to 2.51±0.36 ng/g for tyramine andfrom 0.96±0.31 ng/g to 2.39±0.64 ng/g for tyrosine). The increase inganglionic morphine levels, after tyrosine and tyramine exposure,occurred gradually over the 60 minute incubation period (FIG. 16B).About 7 percent of tyrosine or tyramine appears to be converted tomorphine under these in vitro conditions. Blank runs between morphineHPLC determinations as well as running negative tissue controls, e.g.,mantle, did not reveal a morphine residue with HPLC coupled RIA.Analysis of the marine water and various chemicals used in the protocolalso demonstrated a lack of morphine.

Ganglia treated with quinidine and tyramine exhibited lesstyramine-induced morphine production than the levels observed withganglia treated with tyramine only (FIG. 17). This inhibition ofmorphine production was quinidine concentration dependent (FIG. 17).Likewise, ganglia treated with AMPT and tyrosine exhibited lesstyrosine-induced morphine production than the levels observed withganglia treated with tyrosine only (FIG. 18). This inhibition ofmorphine production was AMPT concentration dependent (FIG. 18). Exposureto either enzyme inhibitor alone did not significantly reduce morphinelevels below the level of non-exposed ganglia (FIGS. 17 and 18).Exposure of pedal ganglia to both enzyme inhibitors, however, reducedganglionic morphine levels significantly (0.23±0.16 ng/g wet weight±SEM;P<0.01) below that of controls (0.99 and 0.92 ng/g wet weight,respectively), indicating that both pathways were workingsimultaneously, compensating for the other's inhibition.

To examine the tyramine to dopamine step, dopamine (DA) levels inganglia and hemolymph were examined before and after tyramine additionfollowed by CYP2D6 inhibition by quinidine. Tyramine injectionsignificantly increased ganglionic (4.98±0.27 μg/g to 9.17±1.21 μg/g wetweight; P<0.01) and hemolymph (10.13±1.24 μg/mL to 16.47±1.28 μg/mL,P<0.05) DA levels (FIGS. 19 and 20). The ganglionic and hemolymph DAlevel increases were blocked by quinidine exposure, demonstrating thatthe CYP2D6 enzyme was mediating this step. Ganglia were also exposed toTHP, reticuline, DA, or codeine. Each resulted in significantly enhancedmorphine levels, a phenomenon that was also significantly blocked byquinidine exposure, again demonstrating a role for CYP2D6 in the secondpart of the morphine biosynthetic pathway (FIGS. 15 and 20).

A molecular analysis was performed to confirm the pharmacologicalevidence for the involvement of CYP2D6 in ganglia. Briefly, RT-PCR wasused to amplify a 282 bp fragment from Mytilus tissue. The sequence ofthis fragment was found to be about 94 percent similar to the humancytochrome P450, family 2, subfamily D, polypeptide 6 mRNA sequence setforth in GenBank accession number M20403 (FIG. 21).

In in vivo experiments, animals were injected with either tyrosine (10⁻⁵M) or tyramine (10⁻⁵ M) in their foot. One hour after injection, gangliawere dissected and extracted for morphine analysis. Both precursorssignificantly enhanced ganglionic morphine levels compared with controlvalues (2.46±0.22 ng/g wet weight for tyrosine and 3.28±0.45 ng/g fortyramine compared to controls 1.02±0.24 ng/g; P<0.001; FIG. 22).Statistical significance was not be achieved at the 10⁻⁷ to 10⁻⁶ Mconcentrations, but was achieved at the 10⁻⁵ M concentration (FIG. 22).Additionally, 20 animals per drug protocol were injected via the footwith either AMPT (10⁻⁴ M) or quinidine (10⁻⁴ M). These animals did notexhibit any change in morphine levels even when both AMPT and quinidinewere co-administered. This indicates that basal morphine levels werebeing maintained via morphine storage, or the inhibitors did not reachthe ganglia. Compared to controls injected with saline, the tyrosine andtyramine animals exhibited a significant decrease in ganglionic morphinelevels when the respective enzyme inhibitors were topically applied tothe pedal ganglia of intact animals after they were injected with therespective amino acids in the foot (decrease of 30 and 25 percent,respectively; P<0.01; FIG. 22). In this regard, it was estimated thatonly 1-2 percent of the injected amino acids were directed to morphinebiosynthesis.

Taken together, these results demonstrate that tyrosine and tyramineare, in part, being converted to dopamine then morphine, and that thisprocess can be inhibited by altering either or both CYP2D6 or tyrosinehydroxylase. This process appears to be dynamic in that the inhibitionof one pathway allows the other to continue with morphine synthesis.Moreover, dopamine and morphine synthesis appear to be coupled (FIG.15). In particular, these results demonstrate that morphine biosynthesiscan occur by way of tyrosine and/or tyramine, making it very likely thatmorphine synthesis occurs regardless of circumstances. As demonstrated,neither AMPT or quinidine when administered alone reduced endogenousmorphine levels below that of controls suggesting the presence of astorage pool of morphine. Co-administration of AMPT and quinidinereduced endogenous morphine levels below that of controls. These resultsindicate that if one pathway is blocked, the overall pathway continuedbecause the other complementary pathway to dopamine remains functional.This coupling to dopaminergic processes can have biomedicalimplications. For example, the DA component can modulate excitatorystates, including rage, whereas the morphinergic component can result ina calming action associated with relaxation and reward. This associationcan explain the calming effect that follows excitatory emotional states.

Example 8 Use of Low Dose Morphine

The following experiments were performed to evaluate the ability of lowdoses of morphine to exert biological effects. Whole Mytilus animalswere treated with saline or morphine (10⁻⁶ to 10⁻¹⁰ M) by injection.After a five minute incubation, hemolymph was collected and incubatedwith LPS (1 μg/mL). Cells from animals pre-treated with morphine from10⁻⁷ to 10⁻¹⁰ M did not exhibit a reduction in the level of cellactivation that was observed with LPS-treated cells obtained fromsaline-treated animals (FIG. 23). Cells from animals pre-treated withmorphine (10⁻⁶ M) exhibited 19.3±3.8 percent activation. Thus, lowerdoses of morphine did little to alter the LPS stimulatory action onimmunocytes when administered in an almost concomitant manner.

Whole Mytilus animals were treated daily with saline or morphine (10⁻⁶to 10⁻¹⁰ M) by injection for 4 days. After a five minute incubation,hemolymph was collected and incubated with LPS (1 μg/mL). Cells fromanimals pre-treated with morphine from 10⁻⁷ to 10⁻⁹ M exhibited areduction in the level of cell activation that was observed withLPS-treated cells obtained from saline-treated animals (FIG. 24). Cellsfrom animals pre-treated with morphine (10⁻¹⁰ M) did not exhibit areduction in the level of cell activation. Thus, lower doses of morphine(e.g., 10⁻⁷ to 10⁻⁹ M) can limit excitatory activations and possiblyreduce pre-existing activation when given over time.

100 healthy Mytilus animals were treated with 10 U/mL of TNF-α byinjection. After 4 days, about 20 percent of the TNF-treated animalsdied (FIG. 25). Pre-treatment for four days with daily injections ofmorphine at a low dose (10⁻⁷ M) reduced the number of animals that died(FIG. 25). These results demonstrate that repeated administration of lowdoses of morphine can protect against TNF-α-induced death by apparentlyreducing the level of TNF-α-induced inflammation within the animals.

In another experiment, COS-1 cells were transfected with nucleic acidthat directs expression of a human mu3 opiate receptor. The stablytransfected cells were then incubated with morphine, and the amount ofnitric oxide (NO) released from the cells was measured amperometrically.Cells treated with 10⁻⁷ M and 10⁻⁸ M of morphine released 9±2 nM and18±3 nM of NO, respectively, within 2 minutes of morphine addition.These results demonstrate that the mu3 opiate receptor mediates morphinecoupled no release.

In another experiment, human saphenous veins were treated with morphineand assessed for NO release. Tissue treated with 2×10⁻⁷ M morphinereleased 7±2 nM of NO. When pre-treated with 10⁻⁶M of CTOP, an opiatereceptor inhibitor specific for mu receptors, 10 minutes before addingmorphine, no NO release was detected. These results demonstrate that theNO release was mediated via an opiate receptor.

Mytilus animals were divided into three groups. The first group was acontrol group with each animal being untreated prior to receiving asingle injection of saline. The second and third groups of animalsreceived daily injections of 1 μM and 0.01 μM of morphine, respectively,via the foot. Two hours post-injection, pedal ganglia were obtained fromthe animals and assessed for mu3 opiate receptor binding densities. Thisexperiment was repeated 5 times, each with a separate set of Mytilusanimals.

Treatment with 1 μM of morphine resulted in reduced mu3 opiate receptorbinding, while treatment with 0.01 μM of morphine did not (FIG. 27).These results demonstrate that low dose morphine is effective withoutaltering binding site densities.

45 Mytilus animals were divided into three groups. The first group was acontrol group with each animal receiving a mock injection of saline. Thesecond and third groups of animals received daily injections of 1 μM and0.01 μM of morphine, respectively, via the foot for up to four days. Twohours post-injection, NO release was measured. This experiment wasrepeated 5 times, each with a separate set of Mytilus animals.

Animals receiving 1 μM morphine exhibited NO release on day one (FIG.28). The level of NO release for animals receiving 1 μM morphine forfour days, however, was substantially lower than the level of NO releaseobserved after one day of treatment with 1 μM morphine. When challengedwith 10 μM morphine, animals receiving 1 μM morphine for four daysexhibited about half the amount of NO release observed with animalsreceiving 1 μM morphine for one day. These results demonstrate thatanimals receiving repeated administrations of 1 μM morphine developtolerance to morphine. Animals receiving 0.01 μM morphine exhibited NOrelease on day one at a level similar to that which was also observedafter days two, three, and four (FIG. 28). These results demonstratethat animals receiving repeated administrations of 0.01 μM morphinecontinue to respond to morphine administration without developingdetectable or significant tolerance to morphine.

In another experiment, human SH-SY5Y cells were cultured in 96 wellplates (250,000 cells per well). The cells were continuously exposed to10⁻⁸ M or 10⁻⁶M of morphine sulfate. At various time points (initial and1, 2, and 7 days), the cells were washed in phosphate buffered saline(PBS), placed in 250 μL PBS, and assessed for NO release. NO release wasmeasured using an Apollo-4000 free radical analyzer with a 30 μm probe.The probe was calibrated daily with SNAP. Each assay was performed inquadruplicate. The mean±the standard error were graphed for each timepoint. Control cells remained untreated until being challenged with 10⁻⁶M morphine.

Cells treated with a high morphine dose of 10⁻⁶ M lost their initiallevels of NO release, while cells treated with a low morphine dose of10⁻⁸ M remained capable of releasing NO in response to morphine (FIGS.29 and 30). Cells treated daily with 10⁻⁶ M morphine for six days andgiven 10⁻⁶ M of morphine on the seventh day exhibited 3.4 nM of NOrelease. These results demonstrate that tolerance occurs only at thehigh dose.

Example 9 Morphine-6β-glucuronide Increases mu3 Opiate ReceptorExpression levels

Human blood cells (mononuclear cells and polymorphonuclear cells) wereincubated with 10⁻⁷M morphine-6β-glucuronide (M6G) for 30 minutes andassessed from the relative gene expression level of mu3 opiate receptorsequences using real time RT-PCR (Applied Biosystems 5700 SDS). Inaddition, the cells were either incubated with or without 10⁻⁶M CTOP 10minutes prior to adding M6G.

Both mononuclear cells and polymorphonuclear cells exhibited an increasein mu3 opiate receptor expression when treated with M6G (FIG. 31). Inboth cases, the increase in mu3 opiate receptor expression levels wasblocked by CTOP pre-treatment (FIG. 31). These results demonstrate thatthe increase in mu3 opiate receptor expression levels is mediated byM6G.

Example 10 Morphine Modulates β-Amyloid Metabolism Via Nitric OxideProviding a Protective Mechanism for Morphine in Alzheimer's Disease

The deposition of intracellular and extracellular β-amyloid peptide (Aβ)in the brain is a pathologic feature of Alzheimer's disease (AD), aprevalent neurodegenerative disorder. The following experiments wereperformed to better understand the role of Aβ in causing AD's symptoms.

Methods and Materials

SH-SY5Y human neuroblastoma cells (ATCC, USA) were cultured inDulbecco's modified Eagle's medium/Ham's nutrient mixture (DMEM-F12)(Invitrogen, USA), and HTB-11 human neuroblastoma cells (ATCC, USA) werecultured in Minimum Essential Medium Alpha Medium (MEM-α) (Invitrogen,USA). Cells were kept in a 37° C. incubator (Napco) gassed with 5%CO₂/95% air. All treatments were performed under a sterile hood.

Reverse transcriptase-polymerase chain reaction (RT-PCR) was performedto analyze the effects of Aβ₁₄₂, morphine, and SNAP treatments uponBACE-1 and BACE-2 mRNA expression in SH-SY5Y and HTB-11 cells. After thetreatment time-period, cells were harvested, and total RNA was extractedusing RNeasy RNA Isolation kit (Qiagen) following the manufacturer'sprocedures. Total RNA yield was determined using a SpectrophotometerRNA/DNA calculator (Pharmacia Biotech). Total RNA concentration was thenstandardized for semi-quantitative RT-PCR, which was carried out in aGeneamp Thermocycler PCR System 9700 (P.E. Applied Biosystems). Primersused for PCR were as follows: Forward 5′-TGACTGGGAACACCCCATAACT-3′ (SEQID NO:7) and reverse 5′-CGAGCGCCTCAGTGTTACTCT-3′ (SEQ ID NO:8) forBACE-1, and forward 5′-AGCCATCCTCCTTGTCTTAATCG-3′ (SEQ ID NO:9) andreverse 5′-TCTGGCGGAAAATAACCTCAA-3′ (SEQ ID NO:10) for BACE-2. Theexpected product length was 556 bp for both primer sets. PCR productsand a 100 bp DNA marker were then loaded in a 2% agarose gel stainedwith ethidium bromide. Gel electrophoresis was performed using apower-supply (E-C Apparatus Corp.) set at 110V with constant amperagefor 1.5 hours. Gels were then photographed using a Gel DocumentationSystem (UVP), and bands analyzed using Gel-Pro Analyzer(MediaCybernetics) on a P4 Windows machine. Expression levels werestandardized to a reference gene, cyclophilin, using the followingprimers: forward 5′-TTTCGTGCTCTG-AGCACTGG-3′ (SEQ ID NO:11) and reverse5′-CTTGCCATTCCTGGACCCAA-3′ (SEQ ID NO:12).

The production of NO in SH-SY5Y cells was detected using the Apollo 4000Free Radical Analyzer (World Precision Instruments). SH-SY5Y cells weretrypsinized and cultured in a 6-well plate for 48 hours. An L-shapedamperometric NO-specific probe was connected to the Apollo Analyzer andcalibrated using a SNAP+CuCl₂ solution, which releases calculableamounts of NO. Cells were pretreated with 10 and 25 μM Aβ for 30 minutesand 24 hours. At the end of the treatment time-point, the media wasremoved and replaced with PBS warmed in a 37° C. bath, which isnon-reactive with the probe. The probe was inserted about 1.5 mm abovethe cells and allowed to equilibrate for 5 minutes. Then,morphine-6β-gluconuride (M6G) was added to each plate at a concentrationof 1 μM. M6G attaches to G-protein-coupled mu3 receptors on SH-SY5Ycells, stimulating release of Ca⁺² ions which activate cNOS (Cadet etal., Frontiers in Bioscience, 9:3176-86 (2004)), thereby normallyreleasing constitutive NO from neuroblastoma cells within minutes. Theprobe was monitored in real-time for the production of NO “spikes.” NOdata was recorded using Free Radical Analyzer (World PrecisionInstruments). Cells were then discarded.

Aβ₁₋₄₂, nitro-L-arginine methyl ester (L-NAME), ethidium bromide, andtrypsin-EDTA were purchased from Sigma-Aldrich, USA. 0.1 Mdithiothreitol (DTT), 10× Polymerase Chain Reaction (PCR) buffer,Superscript reverse-transcription enzyme, TAQ polymerase, 50 μM MgCl₂,5× First Strand Buffer, the custom PCR primers, and random primers werepurchased from Invitrogen, USA, and stored at −20° C. Phosphate-bufferedsaline was also purchased from Invitrogen, USA, and stored at roomtemperature. Nucleotides (dNTPs) were purchased from Amershar PharmaciaBiotech, USA, and stored at 25 μM concentration at −20° C. RNeasy RNAIsolation reagents and columns were purchased from Qiagen, USA. A stocksolution of the Aβ₁₋₄₂ peptide was prepared at 1 mM concentration andkept frozen at −20° C. Electrophoresis-grade agarose was purchased fromFisher Biotech, USA, and stored at room temperature.S-Nitroso-N-acetyl-D, L-penicillamine (SNAP) used for both celltreatment and NO detector calibration was purchased from World PrecisionInstruments, USA.

Results

Untreated HTB-11 cells constitutively express BACE-1 and BACE-2 mRNA.Morphine exposure to these cells down regulates the expression of BACE-1after 24 hours in a concentration dependent manner (1 μM dosage having agreater effect than 5 μM, 44% as compared to 18%; FIG. 32).Simultaneously, morphine up regulates the expression of BACE-2expression in HTB-11 cells, an effect enhanced in the presence of Aβ₁₋₄₂(FIG. 33). Since BACE-1 promotes production of Aβ₁₋₄₂ and BACE-2inhibits it, morphine can be neuroprotective since morphine modulationof the BACE enzymes would decrease Aβ₁₋₄₂ production. Morphine's effectson both BACE-1 and BACE-2 expression were blocked by naloxone (FIG. 34),verifying that the neuroprotective action of morphine is directlyrelated to its binding to the mu3 opiate receptor.

One of endogenous morphine's primary physiological effects is cNOSderived NO release via mu3 opiate receptor subtype coupling. Todetermine whether morphine's neuroprotective effects on the Aβ pathwaywere NO dependent, HTB-11 cells were treated with L-NAME, a cNOSinhibitor. L-NAME significantly blocked the effects of morphine (FIG.35), indicating that NO release is involved in morphine'sneuroprotective moderation of BACE-1 and -2. HTB-11 cells were exposedto SNAP, a NO donor, and then analyzed for BACE expression levels. After4- and 24-hour exposures, cells treated with SNAP exhibited reducedBACE-1 expression in a concentration dependent manner similar to thatobserved with morphine, which also was enhanced in the presence ofAβ₁₋₄₂ (FIGS. 36 and 37). SNAP also up regulated in a concentrationdependent manner BACE-2 expression at both the 4- and 24-hour timepoints(FIGS. 38 and 39), as did morphine. In the presence of Aβ₁₋₄₂, SNAPdose-dependently increased BACE-2 expression (FIGS. 38 and 39).

To verify the semi-quantitative accuracy of the RT-PCR procedures and toexplore whether the effects of SNAP on BACE expression occur earlierthan four hours, gene expression of BACE-1 and BACE-2 was analyzed in anadditional mRNA expression experiment for two hours (FIG. 40). BACE-1and BACE-2 expression levels were altered in cells treated with SNAP fortwo hours with BACE-1 expression being down-regulated and with BACE-2expression being up-regulated. The expression of the reference gene3-actin was not affected.

SH-SY5Y neuroblastoma cells normally release NO via cNOS in response toapplication of either morphine or its metabolite, M6G. To determinewhether Aβ₁₋₄₂ disrupts this process, SH-SY5Y cells were pre-treatedwith varying concentrations of Aβ₁₋₄₂ for 1 hour. Following the additionof M6G, the Aβ₁₋₄₂-treated cells exhibited a dose-dependent decrease inNO release, demonstrating that Aβ₁₋₄₂ is dose-dependently inhibiting therelease of constitutive NO (FIG. 41). Pretreatment with L-NAME (10⁻⁴ M),a cNOS inhibitor, for 4 minutes also prevented M6G-induced release ofNO, verifying that M6G was inducing release of NO through cNOS given therapid time course of the coupling (FIG. 41; panel G). The reduction ofM6G-induced NO release after Aβ₁₋₄₂ treatment suggests that Aβ₁₋₄₂ iseither (a) directly inhibiting the activation of cNOS or (b) interferingwith the binding of M6G to the mu3 opiate receptor, both of which wouldpotentially impact the level of basal NO in the AD-afflicted humanbrain. Furthermore, SH-SY5Y cells release NO at a low level compared tohuman immune and vascular tissues (3-4 nM compared to 26-29 nM whentreated with morphine at 10⁻⁶ M).

These results demonstrate that morphine, in a concentration and timedependent manner, up regulates BACE-2 expression while simultaneouslydown regulating BACE-1 expression. This phenomenon can be blocked bytreating the cells with the opiate receptor antagonist, naloxone. Thismorphine-mediated process is coupled to cNOS-derived NO release, whichwas ascertained by treating the tissue with the NOS inhibitor L-NAME. NOalone can mediate this effect, further substantiating this observation.Additionally, in the presence of Aβ₁₋₄₂, both the morphine and NOeffects are enhanced. Aβ₁₋₄₂ alone appears to have the ability toinhibit cNOS-derived NO release at higher concentrations. A two-wayrelationship between Aβ₁₋₄₂ and morphine/NO appears to exist.Morphine/NO modifies the expression of two polypeptides involved in theproduction of Aβ, down regulating BACE-1 expression and up regulatingBACE-2 expression. In addition, after long-term incubation, Aβ₁₋₄₂appears to enhance the ability of NO to modify BACE expression. Takentogether, morphine, via its coupling to NO, appears to beneuroprotective since it promotes BACE-2 up regulation, which enhancesAβ catabolism, avoiding the effect of Aβ inhibiting NO production.

The results provided herein can support the following pathway regardingthe origin of AD (FIG. 42). Assuming a deficiency of endogenous morphineor other cNOS activators/scavengers, a decrease in levels of basal NOcan occur over time. Reduced NO levels can result in increased BACE-1expression and reduced BACE-2 expression. More BACE-1 then becomesavailable to cleave APP into Aβ, and Aβ levels increase. Aβ can then besecreted out of the cell to aggregate into amyloid plaques, and solubleAβ levels increase within the cell. Internalized Aβ can inhibit NOrelease by the cell, which then can create a vicious cycle causing NOlevels to be further decreased, lessening regulation of the BACE genes,which again increases the production of Aβ. Simultaneously, Aβ canpromote a chronic and progressively increasing inflammatory reaction,initiating both vascular and neural damage. As the pathology of ADcontinues, NO levels can decrease to a point where hypoperfusion of thebrain becomes chronically destructive. In brain cells, oxidative stresscan increase, and neurons can undergo apoptosis. The result can be anoverall decrease in neuronal function, producing memory loss, cognitivedisorders, and other typical symptoms of AD.

Example 11 Modulation of the Ubiquitin-Proteasome Complex Via MorphineCoupled No Release

The following experiments were performed to determine if morphine and NOplay a role in the prevention of cellular stress via protein metabolism.In particular, the following experiments were performed to determine ifmorphine, via stimulating the production of NO, protects neural cells byattenuating the induction of cellular stress and imbalances in proteinmetabolism.

Methods and Materials

The human SK-N-SH neuroblastoma cell line (ATCC #HTB-11) was used as acellular model. Cells were propagated in Minimum Essential Medium alpha(MEMα) with 10% fetal bovine serum (FBS), 2% penicillin/streptomycin,1.5 g/L NaHCO₃, and 1.0 mM pyruvic acid. A 5% CO₂ incubator (NAPCO) at37° C. was used for maintenance of temperature and pH. A trypsin-EDTAsolution (0.25% trypsin, 0.03% EDTA) was used to aspirate and pellet theadherent cells (400 G for 5 minutes at room temperature). When needed,cells were plated in 6- or 12-well plates using a hemocytometer (2×10⁵cells/well in 6-well plates or 1×10⁵ cells/well in 12-well plates).Experimental manipulations were performed under sterile conditions undera lamina airflow hood after cells had adhered to the bottom of theplates.

Compounds were weighed accurately using an atomic balance and elutedwith solvent under sterile conditions. Rotenone (Sigma), a mitochondrialcomplex I inhibitor, and IFNγ (Endogen) were used to stimulate oxidativeand inflammatory stress. Morphine sulfate (Sigma) was obtained insolution along with naloxone (a mu3 opiate receptor antagonist) andL-NAME (an NO synthase inhibitor).

Cell viability was determined via Trypan blue exclusion. An invertedlight microscope (Nikon) with a phase-contrast filter was used toobserve the cells. Pictures of the cells were taken via a digital camera(Optronix) attached to the microscope ocular. Images were uploaded ontoImagePro Plus Software (Applied Biosystems), where cell viability wascalculated based on both the number of cells covering the field and thecells that present as dead from the stain. In addition, ImagePro Pluswas used for determining cellular morphology. The area and perimeterwere found and used in the formula (4π*Area)/(Perimeter)², resulting ina value between 0 (a straight line) and 1 (a perfect circle).

Isolation of total RNA was performed via RNA MiniPrep Kit (QIAGEN).Concentration of RNA was determined via GeneQuant II Spectrophotometer(Pharmacia Biotech) by multiplying A260 value with dilution factor andnucleic acid constant (0.04). Agarose gel electrophoresis (1%) was usedto check for RNA quality.

RT and PCR reactions were performed in GeneAmp PCR System 9700 (AppliedBiosystems) using reverse transcriptase and Taq DNA Polymerase. Forwardand reverse gene specific-primers for various subunits of the 20Sproteasome and immunoproteasome were either obtained from the literatureor designed through Primer Express Software 2.0 (Applied Biosystems)(Table 4). NMDA receptor subunit primers were also designed (Table 4).NMDA receptor expression was used as a marker of neurodegenerativedisease.

TABLE 4 Gene Specific Forward and Reverse Primers Forward PrimerReverse Primer Fragment Primer Name (5′→ 3′) (5′→ 3′) Size20S Proteasome CTCGCCTTCAAGTTCCAGCA TGCAGCAGGTCACTGACATC 483 bpLMP7 Subunit (SEQ ID NO: 13) (SEQ ID NO: 14) (β5i) 20S ProteasomeAGAGACCGCTACCGGTGAACC TGCAGCAGGTCACTGACATC 245 bp X Subunit (β5)(SEQ ID NO: 15) (SEQ ID NO: 14) 20S Proteasome AGATACCAACACAACGATATGCTCTCCAAGTAAGTACGAGC 230 bp C2 Subunit (α) (SEQ ID NO: 16)(SEQ ID NO: 17) 20S Proteasome TCAGGTGGTGTTCGTCCATT TTCAAAGCTTTCCTTTAGGG220 bp C3 Subunit (α) (SEQ ID NO: 18) TT (SEQ ID NO: 19) NMDA NR1GATGTCTTCCAAGTATGCGGA GGGAATCTCCTTCTTGACCAG 667 bp Subunit(SEQ ID NO: 20) (SEQ ID NO: 21) NMDA NR2B CCCAGCATTGGCATTGCTGTCCATGATGTTGAGCATTACGGA 394 bp Subunit (SEQ ID NO: 22) (SEQ ID NO: 23)β-actin GTGGGGCGCCCCAGGCACCA CTCCTTAATGTCACGCACGA 557 bp Reference Gene (SEQ ID NO: 24) TT (SEQ ID NO: 25)

All primers were optimized for annealing temperatures and cycles (Table5). Gel electrophoresis (2% agarose, 0.01% ethidium bromide) wasperformed and analyzed using a UV transilluminator (UVP) and GelProAnalyzer Software (Applied Biosystems). Relative band intensity wasnormalized with β-Actin reference gene.

TABLE 5 Primer Optimization Number Annealing of Primer Name 30 secCycles 20S Proteasome LMP7 Denaturation 57° C. Extension 32 Subunit(β5i) 95° C. 72° C. 20S Proteasome X 30 sec 57° C. 1 min 30 Subunit (β5)20S Proteasome C2 55° C. 35 Subunit (α) 20S Proteasome C3 55° C. 35Subunit (α) NMDA NR1 Subunit 55° C. 35 NMDA NR2B Subunit 65° C. 35β-actin 55° C. 25 Reference Gene

To determine protein concentrations, protein was harvested using thefollowing buffer: 20 mM Hepes (Sigma), 100 mM NaCl (Fisher), 10 mM NaF(Sigma), 1% Triton X-100 (Sigma), 1 mM sodium orthovanadate (Sigma), 10mM EDTA (Sigma), pH 7.4, 0.1% protease inhibitor cocktail (Sigma), andddH₂O. Homogenized samples were spun in an ultracentrifuge at 10,000 Gfor 20 minutes at 4° C. Concentration was determined via the Bradfordassay (Bio-Rad) using bovine serum albumin (BSA, Sigma) for standardcurve. To eliminate any potential pipetting error, samples and standardswere read in triplicate.

Western blotting was performed as follows. SDS-polyacrylamide gels (12%for separation and 5% for stacking) were made using the Mini Trans-BlotCell (Bio-Rad). Based on the concentrations from the proteinconcentration determination assay, the proteins were mixed with 2×Protein Loading Buffer (0.2M DTT) for a final volume of 24 μL.Tris-Glycine Electrophoresis Buffer (1×) was used in SDS-PAGE, andproteins were separated at constant amperage (35 mA).

Subsequent protein transfer to a nitrocellulose membrane involved use ofthe Mini Trans-Blot Cell apparatus run at constant voltage (100V) for 40minutes. Overnight blocking was performed in 5% non-fat dry milkdissolved in TBST buffer at 4° C. Primary and secondary antibodies wereused (Table 6). The primary incubation lasted for 1 hour and wasfollowed by 4 washes with TBST buffer, 10 minutes each. The secondaryincubation lasted for 1 hour and was followed by 3 washes with TBSTbuffer, 15 minutes each.

TABLE 6 Primary and Secondary Antibodies Type Dilution Company PrimaryAntibody 20S Proteasome X Rabbit 1:1000 Affinity (β5) Subunit PolyclonalBioreagents, Inc. Immunoproteasome LMP7 Rabbit 1:1000 Affinity (β5i)Subunit Polyclonal Bioreagents, Inc. Ubiquitin (whole Mouse 1:100 SantaCruz molecule) Monoclonal Biotechnologies, Inc. Secondary Antibody HRPConjugated Anti-Mouse 1:12500 Pierce, Inc. HRP Conjugated Anti-Rabbit1:12500 Sigma-Aldrich, Inc.

Substrate (Pierce) was administered as a means of stimulatingchemiluminescence. Blots were developed and subsequently analyzed forrelative band intensity using Gel-Pro software (Applied Biosystems).

Proteasome function assays were performed in an effort to examine themodulation in levels of protein degradation. Fluorometric assays wereperformed for both the chymotrypsin activity of the 26S proteasome aswell as activity of the whole 20S proteasome using procedures adaptedfrom Kotamraju et al. (Proc. Natl. Acad. Sci. USA, 100:10653-8 (2003)).Excitation at 365 nM and emission at 460 nM of the fluorogenic compound,4-amido-7-methylcoumarin (AMC), were measured using aSpectrofluoroscence Detector (McPherson).

Nitric oxide release was determined upon administration of morphinesulfate to the neuroblastoma cells through the use of the Apollo 4000free radical detector (WPI Sarasota, Fla.). The amperometric NO probewas calibrated based upon a solution of 0.2M CuCl₂ using the NO donor,S-nitroso-N-acetyl-L-penicillamine (SNAP) at concentrations of 10 μM, 20μM, 40 μM, and 80 μM. To make sure morphine was not reacting with theprobe, negative controls were performed using PBS.

All data from cell viability and cell morphology experiments wasnormalized with the standard error of the mean (±SEM). RT-PCR data wasnormalized with β-actin reference gene expression and, subsequently,±SEM from the average obtained over the course of three trials. Westernblotting involved densitometric analysis of band intensity basedexpression of protein. Functional assays for proteasome activity werenormalized with ±SEM for 3 trials. For all experiments, two-variablet-tests were used to compare levels of significance between treatments.

Results

Morphine-stimulated, cNOS-derived NO release occurred in neuroblastomacells (FIGS. 43A and B; peak value of 22.3 nM±0.85, p<0.001 compared tobaseline 2 nM NO, baseline was insignificant when compared to negativecontrols). Naloxone (10⁻⁶ M) and L-NAME (10⁻⁴ M) blocked the NO releaseinduced by 10⁻⁶ M morphine (3.2 nM NO and 0.3 nM NO, respectively).

Rotenone, an agent inducing oxidative stress at the intracellular level,stimulated cellular death when used to treat neuroblastoma cells(LD₅₀=30 nM). Cell viability experiments (FIGS. 44A and B) involved a24-hour pretreatment of morphine sulfate (5 μM) and a subsequent 48-hourincubation with rotenone (30 nM). This resulted in a significant levelof protection (p<0.01) between the rotenone-treated cells, whichexhibited about 45% confluency, and the morphine-pretreated cells, whichexhibited an increase to about 78% confluency.

Cellular morphology was used as an indicator of cell activation and wascalculated via the Form Factor (FF; FF=1 is a round cell that isimmobile and less than 0.7 indicates an elongated adhering cell; FIG.44C). The FF determination demonstrated that morphine produced aneuroprotective effect (cells were round and inactive) against thetoxicity of a 24-hour pretreatment with rotenone (p<0.003; FIG. 44C). Areversal in morphine neuroprotection was achieved via treatments withnaloxone (10 μM), an opiate receptor antagonist, and L-NAME (10 μM), aninhibitor of NO, suggesting that NO was involved with morphine'sprotective action.

The following experiments were designed to determine the molecularevents involved in the protective process. Relative band intensity wasmeasured in arbitrary units (AU) through a computer-assisted imagingsystem. Rotenone was examined for its ability to induce an imbalance inthe expression of a molecular marker for neurodegenerative disease, theNMDA receptor. A rotenone-induced imbalance was observed in the NMDAreceptor (NR1 and NR2B subunits) mRNA expression, and morphine reversedthe imbalance of this indicator. NR1 expression in controls (1.57±0.072AU) was decreased with rotenone to 1.22±0.010 AU (LD₅₀, 30 nM) and1.21±0.028 AU (40 nM; p<0.003 when compared to control) (FIG. 45A).Morphine (5 μM) significantly increased NR1 expression to 1.40±0.056 AUat the LD₅₀ of rotenone (p<0.035). NR2B expression was 0.98±0.02 in thecontrol compared to 1.22±0.08 AU and 1.49±0.02 AU (p<0.001 for bothcompared to control) with 30 nM (LD₅₀) and 40 nM rotenone, respectively(FIG. 45B). A significant decrease in NR2B expression to 1.09±0.06 AUand 1.17±0.04 AU with morphine (5 μM) administration was observed with30 nM and 40 nM of rotenone, p<0.042 and p<0.018, respectively, comparedto rotenone values.

Examination of expression of various subunits of the 20S proteasomeinitially involved testing for mRNA expression of the proteasomalnon-catalytic C2 and C3 alpha subunits as well as catalytic X (β5)subunit since this may be the sight of the effect. No change in thelevel of expression of the alpha subunits was observed.

Regulation of X subunit expression was observed with rotenone andmorphine, indicating neuroprotection (FIG. 46). Morphine increased thelevel of expression of the proteasomal X subunit in a dose dependentmanner at both 4 and 24 hours (p<0.014 at 4 hours, and p<0.009 from0.716±0.015 AU control to 0.868±0.007 AU 5 μM at 24 hours). Morphine (5μM) induced neuroprotection was observed in a dose dependent decrease inX subunit expression, which was significant when compared to treatmentswith rotenone alone (p<0.01 at 4 hours and p<0.012 at 24 hours).

Morphine also stimulated a decrease in mRNA expression of the LMP7immunoproteasome subunit, blocking a slight increase in LMP7 mRNA causedby rotenone (FIG. 47). There was a significant dose dependent decreasein LMP7 expression from the value of the rotenone control at 1 μMmorphine (p<0.026) and to 0.792±0.001 AU at 5 μM morphine (p<0.018).

The data above, regarding mRNA expression, was reinforced throughWestern blotting detection of the X subunit protein levels. Relativeband intensity was measured in arbitrary units through acomputer-assisted imaging system. A time course with morphine treatment(FIG. 48A), indicates a dose dependent significant increase in the levelof expression of the X subunit after morphine administration (1 μM and 5μM) at 24 hours (p<0.01 with 1 μM compared to control and p<0.001 when 5μM). The expression of the X subunit also was examined after concomitantrotenone treatments and morphine exposure (FIG. 48B). The previous mRNAresults (from FIG. 46) were confirmed by changes in protein expression,showing evidence of morphine neuroprotection. The control band intensityof 149 AU increased to 162 AU and 188 AU (1 μM and 5 μM morphine,respectively). Rotenone caused an increase in expression from controlvalue to 182 AU. There was a decrease exhibited in morphine-inducedneuroprotection from 182 AU to 168 AU (1 μM morphine) and subsequentlyto 154 AU (p<0.028).

Experiments determining the modulation of the 20S proteasome andchymotrypsin activity of the 26S proteasome revealed a differentialregulation (FIG. 49). Rotenone decreased the activity of thechymotrypsin functional 26S active site (control value of 236±4.8 μM to221±4.8 μM; p<0.043), whereas morphine (5 μM) increased the controlvalue to 277±8.0 μM (p<0.021). Concomitant treatment of morphine androtenone resulted in a restoring of chymotrypsin activity (p<0.050; FIG.49A). These results demonstrate that, through the increase in 26Schymotrypsin activity, morphine was able to prevent the need forincreased 20S activity by decreasing 20S activity (FIG. 49B) involved indegradation of oxidized and misfolded proteins.

Western blotting revealed that morphine caused a dose dependent increasein the levels of free ubiquitin from the control value of 44 AU to 64 AUand 125 AU (1 μM and 5 μM, respectively). A slight decrease in freeubiquitin, although not significant, was observed with rotenone, whichwas reversed significantly with concomitant administration of morphineand rotenone (p<0.039; FIG. 50). The increase in free ubiquitinexpression—ubiquitin not bound to proteins—stimulated by morphine andsubsequent counteraction against the decreased expression caused byrotenone provides further evidence into the functional value of morphineneuroprotection.

To understand the effects of neuroinflammation, interferon (IFN)γ wasused to simulate an immune response, i.e., proinflammatory. IFNγ did notcause significant cellular death. However, IFNγ did cause changes incellular morphology indicative of the neuroinflammatory stress. A FFincrease from 0.54±0.02 to 0.76±0.02 was observed between control andIFNγ treatments (p<0.014). A decrease in the FF resulted fromconcomitant treatment of both morphine and IFNγ, (FF 0.76±0.02 to0.58±0.01 reveals cellular elongation). A non-significant reversal inmorphine neuroprotection was observed with treatments of naloxone andL-NAME.

IFNγ induction of LMP7 was examined IFN-γ caused an increase in LMP7expression after 24 hours from the control value of 0.29±0.01 to0.93±0.06 (p<0.001), demonstrating neuroinflammatory stress. Morphineblocked the LMP7 increase induced by IFNγ (p<0.034; FIG. 51).

Western blotting for the LMP7, after IFNγ stimulated its expression,revealed that morphine exposure blocked this action at both 36 and 48hours (FIG. 52; p<0.001 for comparison between treatment #2 andtreatment #3; p<0.001 for comparison between treatment #5 and treatment#6).

These results demonstrates that morphine exerts neuroprotective actionsfollowing the administration of rotenone, a compound that initiatescellular oxidative stress thereby inducing a high rate of cell death.Morphine exerts the same protective mechanism in regard to immunetissues activated by IFN-γ, which also exerts deleterious actions.Additionally, morphine was shown to stimulate production of cNOS-derivedNO in neurobastoma cells. Taken together, morphine appears to exert itsprotective actions via constitutive NO release, where NO may act as anantioxidant.

Rotenone, an inhibitor of mitochondrial complex I, stimulates productionof ROS and causes cell death. Cells treated with morphine prior torotenone exposure, exhibit significant amelioration of cell death.Moreover, rotenone was able alter the molecular imbalance between NR1and NR2B subunits, demonstrating that cellular stress or oxidativedamage occurred. Morphine corrected this imbalance.

In regard to the ubiquitin-proteasome complex mRNA and proteinexpression, morphine increased X subunit expression in a dose dependentmanner. Interestingly, rotenone also stimulated an increase in X subunitexpression, which was blocked by morphine, causing a shift in levels ofexpression back to the control. In addition, experiments were performedto test for the expression of LMP7, an immunoproteasome catalyticsubunit. These experiments revealed an increase in LMP7 mRNA expressionthat was blocked by morphine, further signifying morphine inducedneuroprotection not only against oxidative stress, but also inflammatorystress as well.

Proteasome function assays were performed to observe proteasomeenzymatic activity, i.e., actual protein degradation. Rotenone caused anincrease in activity of the 20S proteasome along with a decrease inactivity of the 26S chymotrypsin active site. The decreased 26Schymotrypsin activity was most probably due to increased surfacehydrophobicity of oxidized proteins, preventing intake into the 19Sregulatory particle. Thus, 20S activity increased in order to degradeoxidized proteins. In this regard, morphine stimulated an increase in26S chymotrypsin activity and a decrease in 20S activity. Thisdifferential effect may have been achieved through the nitration ofoxidized proteins. NO may have marked proteins for degradation via thechymotrypsin active site, specific for nitrated proteins. As moreproteins were degraded through the 26S proteasome, there was a decreasein 20S activity because of the reduced quantity of oxidized proteins.Thus, from the proteasome function assays, morphine neuroprotection canbe attributed not only to prevention of ROS, but also through specifictargeting of proteins for degradation at the chymotrypsin active site onthe 26S proteasome.

Examining free ubiquitin, a marker for protein degradation, revealedlower levels after treatment with rotenone because more ubiquitinmolecules were used to mark oxidized proteins. Interestingly, morphinecaused a dose dependent increase in ubiquitin protein expression. Thisimplies that, upon treatment with morphine, fewer proteins were oxidizedand misfolded, further supporting morphine stimulated neuroprotection.

Examination of the LMP7 mRNA and protein expression revealed thatmorphine inhibited the induction of this immunoproteasome subunit.Western blotting revealed that IFN-γ caused both a decrease in Xexpression, as well as an increase in LMP7 expression. This effect wasblocked by morphine after IFN-γ exposure.

Taken together, these results demonstrate that morphine, via NO,produces neuroprotection not just by acting as an antioxidant, but alsoby targeting oxidized and misfolded proteins for degradation via the 26Sproteasome. Thus, endogenous morphine signaling may normally function toovercome cellular stress processes, revealing a new pharmacological wayto treat neurodegenerative disorders.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for inducing nitric oxide release from cells in a mammal,said method comprising administering, to said mammal, a composition inan amount, at a frequency more frequent than once a week, and for aduration longer than one month, wherein said composition comprisesmorphine or morphine-6β-glucuronide, and wherein said amount of saidcomposition results in less than 0.05 mg of said morphine ormorphine-6β-glucuronide being administered to said mammal per kg of bodyweight of said mammal per day.
 2. The method of claim 1, wherein saidcells are immune cells.
 3. The method of claim 1, wherein said mammal isa human.
 4. The method of claim 1, wherein said composition comprises amorphine precursor.
 5. The method of claim 1, wherein said compositionis in the form of a tablet.
 6. The method of claim 1, wherein saidcomposition comprises selenium.
 7. The method of claim 1, wherein saidcomposition comprises L-arginine.
 8. The method of claim 1, wherein saidcomposition comprises a calcium source.
 9. The method of claim 1,wherein said amount of said composition results in less than 0.025 mg ofsaid morphine or morphine-6β-glucuronide being administered to saidmammal per kg of body weight of said mammal per day.
 10. The method ofclaim 1, wherein said amount of said composition results in less than0.01 mg of said morphine or morphine-6β-glucuronide being administeredto said mammal per kg of body weight of said mammal per day.
 11. Themethod of claim 1, wherein said frequency is more frequent than fourtimes a week.
 12. The method of claim 1, wherein said frequency isbetween two and five times a day.
 13. The method of claim 1, whereinsaid frequency is once a day.
 14. The method of claim 1, wherein saidduration is longer than two months.
 15. The method of claim 1, whereinsaid duration is longer than three months.
 16. The method of claim 1,wherein said composition comprises morphine.
 17. The method of claim 1,wherein said composition comprises morphine-6β-glucuronide.
 18. Themethod of claim 1, wherein said composition comprises morphine andmorphine-6β-glucuronide.
 19. A method for inducing nitric oxide releasefrom cells in a mammal, said method comprising administering, to saidmammal, a composition in an amount, at a frequency more frequent thanonce a week, and for a duration longer than one month, wherein saidcomposition comprises thebaine or codeine, and wherein said amount ofsaid composition results in less than 0.05 mg of said thebaine orcodeine being administered to said mammal per kg of body weight of saidmammal per day.
 20. The method of claim 19, wherein said cells areimmune cells.